HK1231384B - Antibody drug conjugates (adcs) with kinesin spindel protein (ksp) - Google Patents

Description

Kinesin Spindle Protein (KSP)-containing Antibody Drug Conjugate (ADC)

Introduction and current state of the art

The present invention relates to active substance conjugates (ADCs) of spindle kinesin inhibitors, active metabolites of these ADCs, methods for preparing these ADCs, uses of these ADCs for treating and/or preventing diseases, and uses of these ADCs for preparing medicaments for treating and/or preventing diseases, particularly hyperproliferative and/or angiogenic diseases, such as cancer. Such treatments can be performed as monotherapy or in combination with other agents or other therapeutic measures.

Cancer is the result of uncontrolled cell growth in a wide variety of tissues. In many cases, new cells infiltrate existing tissues (invasive growth) or metastasize to distant organs. Cancers arise in a wide variety of organs and often have a tissue-specific course. The term cancer is therefore a general term that describes a large number of defined diseases in various organs, tissues, and cell types.

Initially, tumors can be removed with surgery and radiation therapy. Metastatic tumors are usually treated with palliative chemotherapy. The goal is to achieve the best possible combination of improved quality of life and prolonged life expectancy.

Conjugates of binding proteins with one or more active substance molecules are known, in particular in the form of so-called "antibody-drug conjugates" (ADCs), in which an internalizing antibody against a tumor-associated antigen is covalently linked to a cytotoxic agent via a linker ("linker"). After introduction of the ADC into tumor cells and subsequent dissociation of the conjugate, the cytotoxic agent itself or cytotoxic metabolites formed therefrom are released within the tumor cells and can exert their effects directly and selectively therein. Thus, unlike conventional chemotherapy, damage to normal tissues can be kept within significantly narrower limits [see, for example, JM Lambert, Curr . Opin. Pharmacol. 5 , 543-549 (2005); AM Wu and PD Senter, Nat. Biotechnol. 23 , 1137-1146 (2005); PD Senter, Curr. Opin. Chem. Biol. 13 , 235-244 (2009); L. Ducry and B. Stump, Bioconjugate Chem. 21 , 5-13 (2010)]. Thus, WO2012/171020 describes ADCs in which multiple toxin cluster molecules are attached to an antibody via a polymeric linker. As possible toxic groups, WO 2012/171020 mentions, inter alia, the substances SB 743921, SB 715992 (Ispinesib), MK-0371, AZD8477, AZ3146 and ARRY-520.

The last substances mentioned are so-called kinesin inhibitors. Kinesin (KSP, also known as Eg5, HsEg5, KNSL1, or KIF11) is a kinesin-like motor protein that is essential for the functioning of the bipolar mitotic spindle. Inhibition of KSP leads to mitotic arrest and, over time, to apoptosis (Tao et al., Cancer Cell 2005 Jul 8(1), 39-59). Since the discovery of the earliest cell-penetrating KSP inhibitor, Monastrol, KSP inhibitors have been established as a new class of chemotherapeutic drugs (Mayer et al., Science 286: 971-974, 1999), and they are the subject of numerous patent applications (e.g., WO2006/044825; WO2006/002236; WO2005/051922; WO2006/060737; WO03/060064; WO03/040979; and WO03/049527). However, because KSP only exerts its effects for a short period during mitosis, KSP inhibitors must be present at sufficiently high concentrations during these early stages.

SUMMARY OF THE INVENTION

Against this background, one object of the present invention is to provide substances which can exhibit apoptotic effects after administration at relatively low concentrations and are therefore useful in cancer treatment.

To achieve this objective, the present invention provides a conjugate comprising a binding agent or a derivative thereof and one or more active substance molecules, wherein the active substance molecule is a kinesin inhibitor (KSP inhibitor) linked to the binding agent via a linker L. The binding agent is preferably a binding protein or peptide, particularly preferably a human, humanized, or chimeric monoclonal antibody or antigen-binding fragment thereof, in particular an anti-TWEAKR antibody or antigen-binding fragment thereof or an anti-EGFR antibody or antigen-binding fragment thereof. Particularly preferred are anti-TWEAKR antibodies that specifically bind to amino acid D (D47) at position 47 of TWEAKR (SEQ ID NO:169), in particular the anti-TWEAKR antibody TPP-2090 or the anti-EGFR antibodies cetuximab or nimotuzumab.

The present inventors have discovered a number of possible ways how binding partners can be attached to KSP inhibitors to achieve the above objectives.

According to the present invention, the spindle kinesin inhibitor may have the following substructure I (sub):

in

#a represents the bond to the rest of the molecule;

R ^1a represents H or -(CH ₂ ) _0-3 Z, wherein Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently of one another represent H, NH ₂ , -(CH ₂ CH ₂ O) _0-3 -(CH ₂ ) _0-3 Z' (e.g. -(CH ₂ ) _0-3 Z') or -CH(CH ₂ W)Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ , COOH, -NH-CO-CH ₂ -CH ₂ -CH(NH ₂ )COOH or -(CO-NH-CHY ⁴ ) _1-3 COOH, wherein W represents H or OH, wherein Y ⁴ independently of one another represent linear or branched C _1-6 -alkyl optionally substituted by -NHCONH ₂ , or represent aryl or benzyl optionally substituted by -NH ₂ ;

R ^2a and R ^4a independently of each other represent H, -CO-CHY ⁴ -NHY ⁵ or -(CH ₂ ) _0-3 Z, or R ^2a and R ^4a together (forming a pyrrolidine ring) represent -CH ₂ -CHR ¹⁰ - or -CHR ¹⁰ -CH ₂ -, wherein R ¹⁰ represents H, NH ₂ , COOH, SO ₃ H, SH or OH, and wherein Z represents -H, -OY ³ , -SY ³ , -NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH;

wherein Y ⁴ independently of one another represents linear or branched C _1-6 -alkyl optionally substituted by —NHCONH ₂ or represents aryl or benzyl optionally substituted by —NH ₂ , and Y ⁵ represents H or —CO—CHY ⁶ —NH ₂ , wherein Y ⁶ represents linear or branched C _1-6 -alkyl.

According to the present invention, the kinesin inhibitor can be linked to the binding body via a linker by replacing the hydrogen atom at R ^1a , R ^2a , R ^4a or R ¹⁰ .

The KSP inhibitor (or KSP inhibitors, as usually more than one KSP inhibitor is attached to the conjugate) attached to the conjugate is preferably a compound of the following formula (Ia) or (IIa):

Formula (Ia), and salts, solvates and salts of solvates thereof:

in

R ^1a represents H, -MOD or -(CH ₂ ) _0-3 Z, wherein Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ , -(CH ₂ CH ₂ O) _0-3 -(CH ₂ ) _0-3 Z' (e.g., -(CH ₂ ) _0-3 Z') or -CH(CH ₂ W)Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ , COOH, -NH-CO-CH ₂ -CH ₂ -CH(NH ₂ )COOH or -(CO-NH-CHY ⁴ ) _1-3 COOH, wherein W represents H or OH,

wherein Y ⁴ independently of one another represent linear or branched C _1-6 -alkyl optionally substituted by -NHCONH ₂ , or represent aryl or benzyl optionally substituted by -NH ₂ ;

R ^2a and R ^4a independently represent H, -CO-CHY ⁴ -NHY ⁵ or -(CH ₂ ) _0-3 Z, or R ^2a and R ^4a together (forming a pyrrolidine ring) represent -CH ₂ -CHR ¹⁰ - or -CHR ¹⁰ -CH ₂ -, wherein R ¹⁰ represents H, SO ₃ H, NH ₂ , COOH, SH or OH,

wherein Z represents -H, halogen, -OY ³ , -SY ³ , NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH;

wherein Y ⁴ independently of one another represents linear or branched C _1-6 alkyl optionally substituted by -NHCONH ₂ , or represents aryl or benzyl optionally substituted by -NH ₂ , and Y ⁵ represents H or -CO-CHY ⁶ -NH ₂ , wherein Y ⁶ represents linear or branched C _1-6 -alkyl;

R ^3a represents -MOD or an optionally substituted alkyl, cycloalkyl, aryl, heteroaryl, heteroalkyl or heterocycloalkyl group,

Preference is given to C _1-10 -alkyl, C _6-10 -aryl or C _6-10 -aralkyl, C _5-10 -heteroalkyl, C _1-10 -alkyl-OC _6-10 -aryl or C _5-10 -heterocycloalkyl, which may be substituted by 1-3 –OH groups, 1-3 halogen atoms, 1-3 haloalkyl groups (each having 1-3 halogen atoms), 1-3 O-alkyl groups, 1-3 –SH groups, 1-3 –S-alkyl groups, 1-3 –O-CO-alkyl groups, 1-3 –O-CO-NH-alkyl groups, 1-3 –NH-CO-alkyl groups, 1-3 –NH-CO-NH-alkyl groups, 1-3 –S(O) _n -alkyl groups, 1-3 –SO ₂ -NH-alkyl groups, 1-3 –NH-alkyl groups, 1-3 –N(alkyl) ₂ groups, 1-3 –NH ₂ groups or 1-3 –(CH ₂ ) _0-3 Z', wherein Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ , wherein Y ¹ and Y ² independently of one another represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H, -(CH ₂ ) _0-3 -CH(NHCOCH ₃ )Z', -(CH ₂ ) _0-3 -CH(NH ₂ )Z' or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH (wherein "alkyl" preferably represents C _1-10 -alkyl);

R ^8a represents C _1-10 -alkyl or —(CH ₂ ) _0-2 —(HZ ² ), wherein HZ ² represents a 4- to 7-membered heterocyclic ring having up to 2 heteroatoms selected from N, O and S;

HZ represents a monocyclic or bicyclic heterocycle which may be substituted by one or more substituents selected from halogen, C _1-10 -alkyl which may be optionally substituted by halogen, C _6-10 -aryl or C _6-10 -aralkyl;

Where –MOD represents –(NR ¹⁰ )n-(G1)o-G2-H, where

R ¹⁰ represents H or C ₁ -C ₃ -alkyl;

G1 represents –NHCO-, -CONH- or (wherein, if G1 represents –NHCO- or , R ¹⁰ does not represent NH ₂ );

n is 0 or 1;

o is 0 or 1; and

G2 represents a straight-chain and/or branched hydrocarbon radical having 1 to 10 carbon atoms and may be interrupted one or more times by one or more of the groups -O-, -S-, -SO-, SO ₂ , -NR ^y -, -NR ^y CO-, CONR ^y -, -NR ^y NR ^y -, -SO ₂ NR ^y NR ^y -, -CONR ^y NR ^y - (wherein R ^y represents H, phenyl, C ₁ -C ₁₀ -alkyl, C ₂ -C ₁₀ -alkenyl or C ₂ -C ₁₀ -alkynyl, each of which may be substituted by -NHCONH ₂ , -COOH, -OH, -NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid), -CO-, -CR ^x =NO- (wherein R x represents H, C ₁ -C ₃ -alkyl or phenyl), wherein the hydrocarbon chain, including side chains, if present, may be interrupted one or more times by -NHCONH ₂ , -COOH, -OH, -NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid substitution, wherein the group -MOD preferably has at least one group -COOH;

wherein the kinesin inhibitor is attached to the linker via substitution of a hydrogen atom at R ^1a , R ^2a , R ^3a , R ^4a , R ^8a or R ¹⁰ or optionally via one of the substituents of HZ, in particular via R ^1a , R ^2a , R ^3a , R ^4a or R ¹⁰ .

Formula (IIa), and salts, solvates and salts of solvates thereof:

in

_X1 represents N, _X2 represents N and _X3 represents C; or

_X1 represents N, _X2 represents C and _X3 represents N; or

_X1 represents CH or CF, _X2 represents C and _X3 represents N; or

_X1 represents NH, _X2 represents C and _X3 represents C; or

_X1 represents CH, _X2 represents N and _X3 represents C

(wherein X ₁ represents CH, X ₂ represents C and X ₃ represents N is preferred);

R ¹ represents H, –L-#1, –MOD or -(CH ₂ ) _0-3 Z, wherein Z represents -H, -NHY ³ , -OY ³ , -SY ³ , halogen, -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ , -(CH ₂ CH ₂ O) _0-3 -(CH ₂ ) _0-3 Z' (e.g., -(CH ₂ ) _0-3 Z') or -CH(CH ₂ W)Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, NH ₂ , SO ₃ H, COOH, -NH-CO-CH ₂ -CH ₂ -CH(NH ₂ )COOH or -(CO-NH-CHY ⁴ ) _1-3 COOH, wherein W represents H or OH,

wherein Y ⁴ independently of one another represent linear or branched C _1-6 -alkyl optionally substituted by -NHCONH ₂ , or represent aryl or benzyl optionally substituted by -NH ₂ ;

R ² represents –L-#1, H, -MOD, -CO-CHY ⁴ -NHY ⁵ or -(CH ₂ ) _0-3 Z, or R ² and R ⁴ together (forming a pyrrolidine ring) represent –CH ₂ -CHR ¹⁰ - or -CHR ¹⁰ -CH ₂ -, wherein R ¹⁰ represents L-#1, H, NH ₂ , SO ₃ H, COOH, SH or OH;

wherein Z represents -H, halogen, -OY ³ , -SY ³ , NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH;

wherein Y ⁴ independently of one another represents linear or branched C _1-6 alkyl optionally substituted by -NHCONH ₂ , or represents aryl or benzyl optionally substituted by -NH ₂ , and Y ⁵ represents H or -CO-CHY ⁶ -NH ₂ , wherein Y ⁶ represents linear or branched C _1-6 -alkyl;

R ⁴ represents –L-#1, H, -CO-CHY ⁴ -NHY ⁵ or -(CH ₂ ) _0-3 Z,

wherein Z represents -H, halogen, -OY ³ , -SY ³ , NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH;

wherein Y ⁴ independently of one another represents linear or branched C _1-6 alkyl optionally substituted by -NHCONH ₂ , or represents aryl or benzyl optionally substituted by -NH ₂ , and Y ⁵ represents H or -CO-CHY ⁶ -NH ₂ , wherein Y ⁶ represents linear or branched C _1-6 -alkyl;

or R ² and R ⁴ together (forming a pyrrolidine ring) represent –CH ₂ -CHR ¹⁰ - or –CHR ¹⁰ -CH ₂ -, wherein R ¹⁰ represents L-#1, H, NH ₂ , SO ₃ H, COOH, SH or OH;

A represents CO, SO, SO ₂ , SO ₂ NH or CNNH;

R ³ represents –L-#1, –MOD or optionally substituted alkyl, cycloalkyl, aryl, heteroaryl, heteroalkyl, heterocycloalkyl, preferably –L-#1 or C _1-10 -alkyl, C _6-10 -aryl or C _6-10 -arylalkyl, C _{5-10 -heteroalkyl, C 1-10} -alkyl-OC _6-10 -aryl or C _5-10 -heterocycloalkyl, which may be substituted by 1-3 –OH groups, _1-3 halogen atoms, 1-3 haloalkyl groups (each having 1-3 halogen atoms), 1-3 O-alkyl groups, 1-3 –SH groups, 1-3 -S-alkyl groups, 1-3 -O-CO-alkyl groups, 1-3 -O-CO-NH-alkyl groups, 1-3 -NH-CO-alkyl groups, 1-3 -NH-CO-NH-alkyl groups, 1-3 -S(O) _n -alkyl groups, 1-3 -SO ₂ -NH-alkyl, 1-3 -NH-alkyl, 1-3 -N(alkyl) ₂ groups, 1-3 -NH ₂ groups or 1-3 -(CH ₂ ) _0-3 Z groups, where Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ , where Y ¹ and Y ² independently of one another represent H, NH ₂ or -(CH ₂ ) _0-3 Z ', and Y ³ represents H, -(CH ₂ ) _0-3 -CH(NHCOCH ₃ )Z ', -(CH ₂ ) _0-3 -CH(NH ₂ )Z ' or -(CH ₂ ) _0-3 Z ', where Z ' represents H, SO ₃ H, NH ₂ or COOH (wherein "alkyl" preferably represents C _1-10 -alkyl);

R ⁵ represents -L-#1, H, NH ₂ , NO ₂ , halogen (especially F, Cl, Br), -CN, CF ₃ , -OCF ₃ , -CH ₂ F, -CH ₂ F, SH or -(CH ₂ ) _0-3 Z, wherein Z represents -H, -OY ³ , -SY ³ , halogen, NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH;

^R6 and ^R7 independently of one another represent H, cyano, (optionally fluorinated) _C1-10 -alkyl, (optionally fluorinated) _C2-10 -alkenyl, (optionally fluorinated) _C2-10 -alkynyl, hydroxy, _NO2 , _NH2 , COOH or halogen (especially F, Cl, Br),

R ⁸ represents (optionally fluorinated) C _1-10 -alkyl, (optionally fluorinated) C _2-10 -alkenyl, (optionally fluorinated) C _2-10 -alkynyl, (optionally fluorinated) C _4-10 -cycloalkyl or –(CH ₂ ) _0-2 -(HZ ² ), wherein HZ ² represents a 4- to 7-membered heterocycle having up to 2 heteroatoms selected from N, O and S, wherein each of these groups may be substituted by –OH, CO ₂ H or NH ₂ or –L-#1;

wherein one or none of the substituents R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁸ and R ¹⁰ represents or (in the case of R ⁸ ) contains -L-#1,

L represents a linker and #1 represents a bond to the conjugate or a derivative thereof,

Where –MOD represents –(NR ¹⁰ )n-(G1)o-G2-H, where

R ¹⁰ represents H or C ₁ -C ₃ -alkyl;

G1 represents –NHCO-, -CONH- or (wherein, if G1 represents –NHCO- or , R ¹⁰ does not represent NH ₂ );

n is 0 or 1;

o is 0 or 1; and

G2 represents a straight-chain and/or branched hydrocarbon radical having 1 to 10 carbon atoms and may be interrupted one or more times by one or more of the groups -O-, -S-, -SO-, SO ₂ , -NR ^y -, -NR ^y CO-, CONR ^y -, -NR ^y NR ^y -, -SO ₂ NR ^y NR ^y -, -CONR ^y NR ^y - (wherein R ^y represents H, phenyl, C ₁ -C ₁₀ -alkyl, C ₂ -C ₁₀ -alkenyl or C ₂ -C ₁₀ -alkynyl, each of which may be substituted by -NHCONH ₂ , -COOH, -OH, -NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid), -CO-, -CR ^x =NO- (wherein R x represents H, C ₁ -C ₃ -alkyl or phenyl), wherein the hydrocarbon chain, including side chains, if present, may be interrupted one or more times by -NHCONH ₂ , —COOH, —OH, —NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid, wherein the group —MOD preferably has at least one group —COOH.

The conjugates of the present invention may have a chemically labile linker, an enzyme-labile linker, or a stable linker. Particularly preferred are stable linkers and linkers cleavable by cathepsins.

The present invention also provides methods for preparing the conjugates of the present invention, as well as precursors and intermediates used in the preparation.

The preparation of the conjugate of the present invention generally comprises the following steps:

(i) preparing a linker precursor which optionally carries a protecting group and has a reactive group capable of coupling to the conjugate;

(ii) coupling the linker precursor to an optionally protected derivative of a low molecular weight KSP inhibitor (preferably a KSP inhibitor having substructure I (sub), particularly preferably a KSP inhibitor of formula (Ia), in particular formula (IIa), wherein in these formulas there is no bond to the linker) to produce a reactive KSP inhibitor/linker conjugate optionally with a protecting group;

(iii) removing any protecting groups present in the KSP inhibitor/linker conjugate, and

(iv) conjugating the binder to the KSP inhibitor/linker conjugate to produce the binder/KSP inhibitor conjugate according to the present invention.

Attachment of the reactive group can also be performed after construction of the optionally protected KSP inhibitor/linker proconjugate.

Depending on the linker, succinimide-linked ADCs can be converted after conjugation to open-chain succinamides with favorable stability profiles according to Scheme 26.

As shown above, ^the linker precursor can be coupled to the ^low molecular weight KSP inhibitor by replacing a hydrogen atom at R ^1a , R ^2a , R ^4a or R ¹⁰ in substructure I (sub), R ^1a , R ^2a , R ^3a , R ^4a , R 8a or R 10 in formula (Ia), or R ¹ , R ² , R ³ , R ⁴ , R 5 ^, R ⁸ or R ¹⁰ in formula (IIa) with a linker. Optional functional groups may also be present in protected form during the synthesis steps prior to coupling. These protecting groups are removed by known methods of peptide chemistry prior to the coupling step. Coupling can be performed chemically by various pathways, as exemplified in Schemes 7 to 31 in the Examples. In particular, the low molecular weight KSP inhibitor to be coupled to the linker can be optionally modified, for example by introducing protecting groups or leaving groups to facilitate substitution.

The present invention particularly provides novel low molecular weight KSP inhibitors that can be optionally coupled to conjugates. These KSP inhibitors or their conjugates have the following general formula (IIIa) and salts, solvates, and salts of solvates thereof:

in

_X1 represents N, _X2 represents N and _X3 represents C; or

_X1 represents N, _X2 represents C and _X3 represents N; or

_X1 represents CH or CF, _X2 represents C and _X3 represents N; or

_X1 represents NH, _X2 represents C and _X3 represents C; or

_X1 represents CH, _X2 represents N and _X3 represents C (wherein _X1 represents CH, _X2 represents C and _X3 represents N is preferred);

R ¹ represents H, –L-BINDER, –MOD or –(CH ₂ ) _0-3 Z, wherein Z represents –H, –NHY ³ , –OY ³ , –SY ³ , halogen, –CO-NY ¹ Y ² or –CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ , -(CH ₂ CH ₂ O) _0-3 -(CH ₂ ) _0-3 Z' (e.g., -(CH ₂ ) _0-3 Z') or -CH(CH ₂ W)Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, NH ₂ , SO ₃ H, COOH, -NH-CO-CH ₂ -CH ₂ -CH(NH ₂ )COOH or -(CO-NH-CHY ⁴ ) _1-3 COOH, wherein W represents H or OH,

wherein Y ⁴ independently of one another represent linear or branched C _1-6 -alkyl optionally substituted by -NHCONH ₂ , or represent aryl or benzyl optionally substituted by -NH ₂ ;

R ² represents –L-BINDER, H, -MOD, -CO-CHY ⁴ -NHY ⁵ or -(CH ₂ ) _0-3 Z, or R ² and R ⁴ together (forming a pyrrolidine ring) represent –CH ₂ -CHR ¹⁰ - or -CHR ¹⁰ -CH ₂ -, wherein R ¹⁰ represents L-#1, H, NH ₂ , SO ₃ H, COOH, SH or OH;

wherein Z represents -H, halogen, -OY ³ , -SY ³ , NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH;

wherein Y ⁴ independently of one another represents linear or branched C _1-6 alkyl optionally substituted by -NHCONH ₂ , or represents aryl or benzyl optionally substituted by -NH ₂ , and Y ⁵ represents H or -CO-CHY ⁶ -NH ₂ , wherein Y ⁶ represents linear or branched C _1-6 -alkyl;

R ⁴ represents –L-BINDER, H, -CO-CHY ⁴ -NHY ⁵ or -(CH ₂ ) _0-3 Z,

wherein Z represents -H, halogen, -OY ³ , -SY ³ , NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH;

wherein Y ⁴ independently of one another represents linear or branched C _1-6 alkyl optionally substituted by -NHCONH ₂ , or represents aryl or benzyl optionally substituted by -NH ₂ , and Y ⁵ represents H or -CO-CHY ⁶ -NH ₂ , wherein Y ⁶ represents linear or branched C _1-6 -alkyl;

or R ² and R ⁴ together (forming a pyrrolidine ring) represent –CH ₂ -CHR ¹⁰ - or –CHR ¹⁰ -CH ₂ -, wherein R ¹⁰ represents –L-BINDER, H, NH ₂ , SO ₃ H, COOH, SH or OH;

A represents CO, SO, SO ₂ , SO ₂ NH or CNNH;

R ³ represents –L-BINDER, –MOD or optionally substituted alkyl, cycloalkyl, aryl, heteroaryl, heteroalkyl, heterocycloalkyl, preferably –L-BINDER or C _1-10 -alkyl, C _6-10 -aryl or C _6-10 -arylalkyl, C _{5-10 -heteroalkyl, C 1-10} -alkyl-OC _6-10 -aryl or C _5-10 -heterocycloalkyl, which may be substituted by 1-3 –OH groups, _1-3 halogen atoms, 1-3 haloalkyl groups (each having 1-3 halogen atoms), 1-3 O-alkyl groups, 1-3 –SH groups, 1-3 –S-alkyl groups, 1-3 –O-CO-alkyl groups, 1-3 –O-CO-NH-alkyl groups, 1-3 –NH-CO-alkyl groups, 1-3 –NH-CO-NH-alkyl groups, 1-3 –S(O) _n -alkyl groups, 1-3 –SO ₂ -NH-alkyl, 1-3 -NH-alkyl, 1-3 -N(alkyl) ₂ groups, 1-3 -NH ₂ groups or 1-3 -(CH ₂ ) _0-3 Z groups, where Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ , where Y ¹ and Y ² independently of one another represent H, NH ₂ or -(CH ₂ ) _0-3 Z′, and Y ³ represents H, -(CH ₂ ) _0-3 -CH(NHCOCH ₃ )Z′, -(CH ₂ ) _0-3 -CH(NH ₂ )Z′ or -(CH ₂ ) _{0- 3} Z′, where Z′ represents H, SO ₃ H, NH ₂ or COOH (wherein “alkyl” preferably represents C _1-10 -alkyl);

R ⁵ represents -L-BINDER, H, NH ₂ , NO ₂ , halogen (especially F, Cl, Br), -CN, CF ₃ , -OCF ₃ , -CH ₂ F, -CH ₂ F, SH or -(CH ₂ ) _0-3 Z, wherein Z represents -H, -OY ³ , -SY ³ , halogen, NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH;

R ⁸ represents (optionally fluorinated) C _1-10 -alkyl, (optionally fluorinated) C _2-10 -alkenyl, (optionally fluorinated) C _2-10 -alkynyl, (optionally fluorinated) C _4-10 -cycloalkyl or –(CH ₂ ) _0-2 -(HZ ² ), wherein HZ ² represents a 4- to 7-membered heterocycle (preferably oxetane) having up to 2 heteroatoms selected from N, O and S, wherein each of these groups may be substituted by –OH, CO ₂ H or NH ₂ or –L-BINDER;

wherein L represents a linker and BINDER represents a binder or a derivative thereof, wherein the binder can optionally be linked to a plurality of active substance molecules,

wherein at most one of R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁸ and R ¹⁰ represents an -L- combination;

^R6 and ^R7 independently of one another represent H, cyano, (optionally fluorinated) _C1-10 -alkyl, (optionally fluorinated) _C2-10 -alkenyl, (optionally fluorinated) _C2-10 -alkynyl, hydroxy, _NO2 , _NH2 , COOH or halogen (especially F, Cl, Br),

Where –MOD represents –(NR ¹⁰ )n-(G1)o-G2-H, where

R ¹⁰ represents H or C ₁ -C ₃ -alkyl;

G1 represents –NHCO-, -CONH- or (wherein, if G1 represents –NHCO- or , R ¹⁰ does not represent NH ₂ );

n is 0 or 1;

o is 0 or 1; and

G2 represents a straight-chain and/or branched hydrocarbon radical having 1 to 10 carbon atoms and may be interrupted one or more times by one or more of the groups -O-, -S-, -SO-, SO ₂ , -NR ^y -, -NR ^y CO-, CONR ^y -, -NR ^y NR ^y -, -SO ₂ NR ^y NR ^y -, -CONR ^y NR ^y - (wherein R ^y represents H, phenyl, C ₁ -C ₁₀ -alkyl, C ₂ -C ₁₀ -alkenyl or C ₂ -C ₁₀ -alkynyl, each of which may be substituted by -NHCONH ₂ , -COOH, -OH, -NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid), -CO-, -CR ^x =NO- (wherein R x represents H, C ₁ -C ₃ -alkyl or phenyl), wherein the hydrocarbon chain, including side chains, if present, may be interrupted one or more times by -NHCONH ₂ , —COOH, —OH, —NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid, wherein the group —MOD preferably has at least one group —COOH.

Description of the drawings

Figure 1: Alignment of the TWEAKR cysteine-rich domain (amino acids 34 to 68) from various species. (Numbers indicate amino acid positions in the full-length construct including the signal sequence; SEQ ID NO: 169).

Figure 2: A – Schematic representation of the structure of TWEAKR (SEQ ID NO: 169). This diagram shows the extracellular domain (amino acids 28-80) (SEQ ID NO: 168), including the cysteine-rich domain (36-67), the transmembrane domain – TM (81-101), and the intracellular domain (102-129). TPP-2202 – The complete extracellular domain (28-80) fused to the Fc domain of hIgG1. TPP-2203 – The extracellular domain with N- and C-terminal truncations (34-68) fused to the Fc domain of hIgG1. The disulfide bridges Cys36-Cys49, Cys52-Cys67, and Cys55-Cys64 are indicated by black bars. At the N- and C-termini, TPP-2203 contains two and one additional amino acids, respectively, compared to the unmodified cysteine-rich domain to ensure proper folding. TPP-1984 – has a C-terminally truncated extracellular domain (28-68) fused to a HIS6 tag. All three constructs showed comparable binding to the antibodies according to the invention and PDL-192 (TPP-1104). P4A8 (TPP-1324) only bound to the full-length extracellular domain (TPP-2202).

B - Amino acid sequence of the extracellular domain: It has been published that amino acid 64 is essential for TWEAK ligand binding; and as determined herein, amino acid 47 is essential for binding of the antibody according to the invention.

Figure 3: Interaction of the TWEAKR extracellular domain with antibodies and reference antibodies. Shown are the results of an ELISA using a TWEAKR-Fc fusion protein coating (TPP-2202, 1 µg/ml) and 0.08 µg/ml (open bars) and 0.03 µg/ml (filled bars) biotinylated IgG as a soluble binding partner. Detection was performed using streptavidin-HRP and Amplex Red substrate. Y is "ELISA signal intensity [Rfu]"; X is "test antibody construct": a is "TPP-2090"; b is "TPP-2084"; c is "PDL-192 (TPP-1104)"; d is "P4A8 (TPP-1324)"; e is "P3G5 (TPP-2195)"; f is "136.1 (TPP-2194)"; h is "ITEM1"; i is "ITEM4"; j is mouse isotype control; k is human isotype control. All antibodies examined showed saturated binding at a concentration of 80 ng/ml.

Figure 4: Interaction of the cysteine-rich domain of TWEAKR with antibodies according to the invention and reference antibodies. Shown are the results of an ELISA using a TWEAKR (34-68)-Fc fusion protein coating (TPP-2203, 1 µg/ml) and 0.08 µg/ml (open bars) and 0.3 µg/ml (filled bars) biotinylated IgG as a soluble binding partner. Detection was performed using streptavidin-HRP and Amplex Red substrate. X is the "test antibody construct": a is "TPP-2090"; b is "TPP-2084"; c is "PDL-192 (TPP-1104)"; d is "P4A8 (TPP-1324)"; e is "P3G5 (TPP-2195)"; f is "136.1 (TPP-2194)"; h is "ITEM1"; i is "ITEM4"; j is a mouse isotype control; k is a human isotype control. All antibodies examined showed saturating binding at a concentration of 80 ng/ml.

Figure 5: Interaction of TWEAKR (28-68) with antibodies according to the invention and reference antibodies. Shown are the results of an ELISA using a TWEAKR (28-68)-HIS coating (TTP-1984, 1 µg/ml) and 0.08 µg/ml (open bars) and 0.3 µg/ml (filled bars) biotinylated IgG as soluble binding partners. Detection was performed using streptavidin-HRP and AmplexRed substrate. X is the "test antibody construct": a is "TPP-2090"; b is "TPP-2084"; c is "PDL-192 (TPP-1104)"; d is "P4A8 (TPP-1324)"; e is "P3G5 (TPP-2195)"; f is "136.1 (TPP-2194)"; h is "ITEM1"; i is "ITEM4"; j is a mouse isotype control; k is a human isotype control. All antibodies examined showed saturating binding at a concentration of 80 ng/ml.

Figure 6: A – Alanine scanning of the cysteine-rich domain. TWEAKR(34-68)-Fc mutants were analyzed for PDL-192(TPP-1104) (X)- and TPP-2090 (Y)-binding. S37A, R38A, S40A, W42A, S43A, D45A, D47A, K48A, D51A, S54A, R56A, R58A, P59A, H60A, S61A, D62A, F63A, and L65A mutants were expressed in HEK293 cells (black diamonds). PFL192(TPP-1104) and TPP-2090 (1 µg/ml) were plated and 8-fold diluted supernatant from HEK293 fermentation broth was added for TWEAKR protein binding. X represents the ELISA strength of the PDL-192/TTP-1104 interaction [Rfu], and Y represents the ELISA strength of the TPP-2090 interaction [Rfu]. TPP-2090 (Y) exhibited reduced binding to the D74A-TWEAKR mutant protein (closed box), and PDL-192 (TPP-1104) (X) exhibited reduced binding to R56A (dashed box).

B – Y are "% binding normalized to wild-type binding signal." 1 is "TPP-2090," 2 is "PDL-192 (TPP-1104)," and 3 is "P4A8 (TPP-1324)." TWEAKR variants were added at 250 ng/ml and detected via anti-HIS HRP. TTP-2090 exhibited less than 5% binding compared to the wild-type construct.

Figure 7: NMR structure of the TWEAKR extracellular domain as published by Pellegrini et al. (FEBS 280:1818-1829). TWEAK binding is dependent on L46 (Pellegrini et al.), TTP-2090 binding is dependent on D47, and PDL-192 binds to R56. PDL-192 binds opposite the TWEAK ligand binding site, while TPP-2090 binds directly to the TWEAK ligand binding site.

Detailed Description of the Invention

The present invention provides a conjugate of a binding body or a derivative thereof and one or more active substance molecules, wherein the active substance molecule is a kinesin inhibitor (KSP inhibitor) connected to the binding body via a linker L.

The conjugate of the present invention can be represented by the following general formula

Wherein BINDER represents a binding entity, preferably an antibody, L represents a linker, KSP represents a KSP inhibitor, m represents a value from 1 to 2, preferably 1, and n represents a value from 1 to 50, preferably 1.2 to 20, particularly preferably 2 to 8. Here, m is the number of KSP inhibitors per linker and n is the average number of KSP inhibitor/linker conjugates per BINDER. The sum of all KSPs present in the conjugate is therefore the product of m and n. KSP-L preferably has the formula (I) or (II) shown above. The binding entity is preferably a binding entity peptide or protein, such as an antibody. In addition, the linker is preferably attached to different amino acids of the binding entity peptide or protein or a derivative thereof. Particularly preferably, it is attached to different cysteine residues of the binding entity.

The following describes combinations that can be used according to the invention, KSP inhibitors that can be used according to the invention, and linkers that can be used according to the invention, which can be used in combination without restriction. In particular, the combinations indicated as preferred or particularly preferred in each case can be used in combination with the KSP inhibitors indicated as preferred or particularly preferred in each case, optionally in combination with the linkers indicated as preferred or particularly preferred in each case.

KSP inhibitors and their conjugates

Low molecular weight KSP inhibitors are known, for example, from WO 2006/044825; WO 2006/002236; WO 2005/051922; WO 2006/060737; WO 03/060064; WO 03/040979; and WO 03/049527.

Typically, KSP inhibitors have the following substructure I(sub):

in

#a represents the bond to the rest of the molecule;

R ^1a represents H or -(CH ₂ ) _0-3 Z, wherein Z represents -H, halogen, NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ , -(CH ₂ CH ₂ O) _0-3 -(CH ₂ ) _0-3 Z' (e.g., -(CH ₂ ) _0-3 Z') or -CH(CH ₂ W)Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ , COOH, -NH-CO-CH ₂ -CH ₂ -CH(NH ₂ )COOH or -(CO-NH-CHY ⁴ ) _1-3 COOH, wherein W represents H or OH,

wherein Y ⁴ independently of one another represent linear or branched C _1-6 -alkyl optionally substituted by -NHCONH ₂ , or represent aryl or benzyl optionally substituted by -NH ₂ ;

R ^2a and R ^4a independently represent H, -CO-CHY ⁴ -NHY ⁵ or -(CH ₂ ) _0-3 Z, or R ^2a and R ^4a together (forming a pyrrolidine ring) represent -CH ₂ -CHR ¹⁰ - or -CHR ¹⁰ -CH ₂ -, wherein R ¹⁰ represents H, NH ₂ , COOH, SO ₃ H, SH or OH,

wherein Z represents -H, -OY ³ , -SY ³ , NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ² or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH,

wherein Y ⁴ independently of one another represents linear or branched C _1-6 -alkyl optionally substituted by —NHCONH ₂ or represents aryl or benzyl optionally substituted by —NH ₂ , and Y ⁵ represents H or —CO—CHY ⁶ —NH ₂ , wherein Y ⁶ represents linear or branched C _1-6 -alkyl.

The following substructures are particularly common: II(sub)

wherein #a, R ^1a , R ^2a , R ^4a have the same meanings as in I(sub) and R ^12a and R ^13a represent H or R ^12a and R ^13a together (forming a piperidine ring) represent –CH ₂ -CHR ¹⁰ - or –CHR ¹⁰ -CH ₂ -, wherein R ¹⁰ represents H, NH ₂ , COOH, SO ₃ H, SH or OH;

wherein Z represents -H, -OY ³ , -SY ³ , NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently of one another represent H, NH ₂ or -(CH ₂ CH ₂ O) _0-3 -(CH ₂ ) _0-3 Z' (e.g. -(CH ₂ ) _0-3 Z'), and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ , COOH or -(CO-NH-CHY ⁴ ) _1-3 COOH, wherein Y ⁴ independently of one another represent linear or branched C _1-6 -alkyl optionally substituted by -NHCONH ₂ , or represent aryl or benzyl optionally substituted by -NH ₂ .

In particular, many KSP inhibitors have substructure II(sub), in which R ^1a , R ^2a , R ^4a , R ^12a and R ^13a represent H.

According to the present invention, KSP inhibitors of substructure I (sub) or substructure II (sub) can be used. KSP inhibitors used according to the present invention also include, for example, Ispins (Cytokinetics/GSK), MK-0731 (Merck), AZD4877 (AstraZeneca), ARRY-520 (Array BioPharma), and ARQ 621 (ArQule).

The preferred KSP inhibitors according to the present invention have the following basic structure and salts, solvates and salts of solvates thereof:

Formula (I):

in

R ^1a represents H, -MOD or -(CH ₂ ) _0-3 Z, wherein Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ , -(CH ₂ CH ₂ O) _0-3 -(CH ₂ ) _0-3 Z' (e.g., -(CH ₂ ) _0-3 Z') or -CH(CH ₂ W)Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ , COOH, -NH-CO-CH ₂ -CH ₂ -CH(NH ₂ )COOH or -(CO-NH-CHY ⁴ ) _1-3 COOH, wherein W represents H or OH,

wherein Y ⁴ independently of one another represent linear or branched C _1-6 -alkyl optionally substituted by -NHCONH ₂ , or represent aryl or benzyl optionally substituted by -NH ₂ ;

R ^2a and R ^4a independently represent H, -CO-CHY ⁴ -NHY ⁵ or -(CH ₂ ) _0-3 Z, or R ^2a and R ^4a together (forming a pyrrolidine ring) represent -CH ₂ -CHR ¹⁰ - or -CHR ¹⁰ -CH ₂ -, wherein R ¹⁰ represents H, SO ₃ H, NH ₂ , COOH, SH or OH,

wherein Z represents -H, halogen, -OY ³ , -SY ³ , NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ₁ and Y ₂ independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH;

wherein Y ⁴ independently of one another represents linear or branched C _1-6 -alkyl optionally substituted by -NHCONH ₂ or represents aryl or benzyl optionally substituted by -NH ₂ , and Y ⁵ represents H or -CO-CHY ⁶ -NH ₂ , wherein Y ⁶ represents linear or branched C _1-6 -alkyl,

R ^3a represents -MOD or an optionally substituted alkyl, cycloalkyl, aryl, heteroaryl, heteroalkyl or heterocycloalkyl group,

Preference is given to C _1-10 -alkyl, C _6-10 -aryl or C _6-10 -aralkyl, C _5-10 -heteroalkyl, C _1-10 -alkyl-OC _6-10 -aryl or C _5-10 -heterocycloalkyl, which may be substituted by 1-3 –OH groups, 1-3 halogen atoms, 1-3 haloalkyl groups (each having 1-3 halogen atoms), 1-3 O-alkyl groups, 1-3 –SH groups, 1-3 –S-alkyl groups, 1-3 –O-CO-alkyl groups, 1-3 –O-CO-NH-alkyl groups, 1-3 –NH-CO-alkyl groups, 1-3 –NH-CO-NH-alkyl groups, 1-3 –S(O) _n -alkyl groups, 1-3 –SO ₂ -NH-alkyl groups, 1-3 –NH-alkyl groups, 1-3 –N(alkyl) ₂ groups, 1-3 –NH ₂ groups or 1-3 –(CH ₂ ) _0-3 Z', wherein Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ , wherein Y ¹ and Y ² independently of one another represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H, -(CH ₂ ) _0-3 -CH(NHCOCH ₃ )Z', -(CH ₂ ) _0-3 -CH(NH ₂ )Z' or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH (wherein "alkyl" preferably represents C _1-10 -alkyl);

R ^8a represents a C _1-10 -alkyl group;

HZ represents a monocyclic or bicyclic heterocycle which may be substituted by one or more substituents selected from halogen, C _1-10 -alkyl which may be optionally substituted by halogen, C _6-10 -aryl or C _6-10 -aralkyl;

Where –MOD represents –(NR ¹⁰ )n-(G1)o-G2-H, where

R ¹⁰ represents H or C ₁ -C ₃ -alkyl;

G1 represents –NHCO-, -CONH- or (wherein, if G1 represents –NHCO- or , R ¹⁰ does not represent NH ₂ );

n is 0 or 1;

o is 0 or 1; and

G2 represents a straight-chain and/or branched hydrocarbon radical having 1 to 10 carbon atoms and may be interrupted one or more times by one or more of the groups -O-, -S-, -SO-, SO ₂ , -NR ^y -, -NR ^y CO-, CONR ^y -, -NR ^y NR ^y -, -SO ₂ NR ^y NR ^y -, -CONR ^y NR ^y - (wherein R ^y represents H, phenyl, C ₁ -C ₁₀ -alkyl, C ₂ -C ₁₀ -alkenyl or C ₂ -C ₁₀ -alkynyl, each of which may be substituted by -NHCONH ₂ , -COOH, -OH, -NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid), -CO-, -CR ^x =NO- (wherein R x represents H, C ₁ -C ₃ -alkyl or phenyl), wherein the hydrocarbon chain, including side chains, if present, may be interrupted one or more times by -NHCONH ₂ , —COOH, —OH, —NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid, wherein the group —MOD preferably has at least one group —COOH.

According to the invention, such spindle kinesin inhibitors can be attached to the linker by substituting a hydrogen atom at R ^1a , R ^2a , R ^3a , R ^4a , R ^8a or R ¹⁰ or optionally via one of the substituents of HZ, in particular via R ^1a , R ^2a , R ^3a , R ^4a or R ¹⁰ .

The substituents of the formula (I) preferably have the following meanings, where these preferred meanings are preferably combined with one another.

R ^1a preferably represents H or -(CH ₂ ) _0-3 Z, wherein Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ , -(CH ₂ CH ₂ O) _0-3 -(CH ₂ ) _0-3 Z' (e.g., -(CH ₂ ) _0-3 Z') or -CH(CH ₂ W)Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ , COOH, -NH-CO-CH ₂ -CH ₂ -CH(NH ₂ )COOH or -(CO-NH-CHY ⁴ ) _1-3 COOH, wherein W represents H or OH,

wherein Y ⁴ independently of one another represent linear or branched C _1-6 -alkyl optionally substituted by -NHCONH ₂ , or represent aryl or benzyl optionally substituted by -NH ₂ ;

R ^2a and R ^4a independently of one another preferably represent H, -COCHY ⁴ -NHY ⁵ or -(CH ₂ ) _0-3 Z, or R ^2a and R ^4a together (forming a pyrrolidine ring) represent -CH ₂ -CHR ¹⁰ - or -CHR ¹⁰ -CH ² -, wherein R ¹⁰ represents H, SO ₃ H, NH ₂ , COOH, SH or OH,

wherein Z represents -H, halogen, -OY ³ , -SY ³ , NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently of one another represent H, NH ₂ or -(CH ² ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH, wherein Y ⁴ independently of one another represent linear or branched C _1-6 -alkyl optionally substituted by -NHCONH ₂ , or represent aryl or benzyl optionally substituted by -NH ₂ , and Y ⁵ represents H or -CO-CHY ⁶ -NH ₂ , wherein Y ⁶ represents linear or branched C _1-6 -alkyl.

R ³ preferably represents an optionally substituted alkyl, aryl, heteroaryl, heteroalkyl, heterocycloalkyl, preferably -L-#1 or C _1-10 -alkyl, C _6-10 -aryl or C _{6-10 -arylalkyl, C 5-10} -heteroalkyl, C _1-10 -alkyl-OC _6-10 -aryl or C _5-10 -heterocycloalkyl, which may be replaced by 1-3 -OH groups, 1-3 halogen atoms, _1-3 haloalkyl groups (each having 1-3 halogen atoms), 1-3 O-alkyl groups, 1-3 -SH groups, 1-3 -S-alkyl groups, 1-3 -O-CO-alkyl groups, 1-3 -O-CO-NH-alkyl groups, 1-3 -NH-CO-alkyl groups, 1-3 -NH-CO-NH-alkyl groups, 1-3 -S(O) _n -alkyl groups, 1-3 -SO ₂ -NH-alkyl, 1-3 -NH-alkyl, 1-3 -N(alkyl) ₂ groups, 1-3 -NH ₂ groups or 1-3 -(CH ₂ ) _0-3 Z groups, wherein Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ , wherein Y ¹ and Y ² independently of each other represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H, -(CH ₂ ) _0-3 -CH(NHCOCH ₃ )Z', -(CH ₂ ) _0-3 -CH(NH ₂ )Z' or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH (wherein "alkyl" preferably represents C _1-10 -alkyl).

R ^8a preferably represents C _1-10 -alkyl.

HZ preferably represents a mono- or bicyclic heterocycle which may be substituted by one or more substituents selected from halogen, C _1-10 -alkyl which may be optionally substituted by halogen, C _6-10 -aryl or C _6-10 -aralkyl.

C _1-10 -alkyl in the context of the present invention (i.e. in the above formulae and in the following formulae) represents a straight-chain or branched alkyl radical having 1 to 10 carbon atoms. Examples which may be mentioned as preferred are: methyl, ethyl, n- propyl, isopropyl, n-butyl, isobutyl, 1-methylpropyl and tert-butyl .

C _6-10 -Aryl- in the context of the invention represents a monocyclic or bicyclic aromatic carbocycle, for example phenyl and naphthyl.

C _6-10 -Aralkyl represents in the present invention a monocyclic aromatic carbocycle to which a C1-C4-alkyl group is attached, for example phenyl. An exemplary C _6-10 -Aralkyl group is benzyl.

_C5-10 -Heteroaryl in the context of the invention represents a monocyclic or bicyclic aromatic heterocycle having a total of 6 to 10 ring atoms, wherein the ring or rings contain one or two ring heteroatoms selected from N, O, S, SO and _SO2 and are linked via a ring carbon atom or optionally a ring nitrogen atom. Examples which may be mentioned are pyridyl, furyl, pyrimidinyl, imidazolyl, thienyl, phenylthio, isoxazolyl, isothiazolyl, 1,2,3-oxadiazolyl, furazanyl, 1,2,3-triazolyl, 1,2,4-triazolyl, pyridazinyl, pyrrolyl, triazinyl, indolyl, quinolyl, quinazolinyl, 1,3-benzodioxolanyl, isoindolyl, indazolyl, 1H-pyrazolo[3,4-d]pyrimidinyl, benzotriazolyl, isoquinolyl, cinnolinyl, phthalazinyl, pteridinyl, naphthyridinyl, benzimidazolinyl, benzothiazolinyl, benzoxazolinyl, 3,4-methylenedioxyphenyl and benzo[6]furanyl.

In the context of the present invention, monocyclic or bicyclic heterocycles are defined as monocyclic or bicyclic heterocycles having a total of 5 to 10 ring carbon atoms, wherein the ring or rings contain one to three ring heteroatoms selected from N, O, S, SO and SO ₂ and are bonded via a ring carbon atom or optionally a ring nitrogen atom. Examples which may be mentioned are piperidinyl, pyrrolinyl, morpholinyl, 3,4-methylenedioxyphenyl and tetrahydrofuranyl.

The halogen atom in the present invention represents F, Cl, Br or I.

The compounds of formula (I) can be linked to the linker in a manner known ^{to those skilled in the art by substitution of a hydrogen atom at R 1a , R 2a , R 4a} , or R ¹⁰ in substructure I (sub) or substructure II (sub) or at R ^1a , R ^2a , R ^3a , R 4a , R ^8a , or R ¹⁰ in HZ in formula (I). Particularly preferred is substitution of a hydrogen atom at R ^1a , R ^2a , R ^3a , R ^4a , or at the pyrrolidine ring formed by R ^2a and R ^4a . This coupling can be carried out chemically by various pathways, as exemplified in Schemes 7 to 31 in the Examples. In particular, the low molecular weight KSP inhibitor to be coupled to the linker can optionally be modified, for example by introducing a protecting group or leaving group to facilitate substitution (such that in this reaction, the leaving group, rather than the hydrogen atom, is replaced by the linker). The KSP inhibitor-linker molecule thus obtained (wherein the linker has a reactive group for coupling to the conjugate) can subsequently be reacted with the conjugate to produce the conjugate conjugate according to the invention. In the experimental part, this procedure is illustrated in an exemplary manner by a number of examples.

R ^1a is preferably H, —COOH, —CONHNH ₂ , —(CH ₂ ) _1-3 NH ₂ , —CONZ″(CH ₂ ) _1-3 NH ₂ and —CONZ″CH ₂ COOH, wherein Z″ represents H or NH ₂ .

R ^2a and R ^4a are preferably H, or R ^2a and R ^4a together (forming a pyrrolidine ring) represent CH ₂ —CHR ¹⁰ — or —CHR ¹⁰ —CH ₂ —, wherein R ¹⁰ represents H.

R ^3a is preferably C _1-10 -alkyl-, which may be substituted by –OH, O-alkyl, SH, S-alkyl, O-CO-alkyl, O-CO-NH-alkyl, NH-CO-alkyl, NH-CO-NH-alkyl, S(O) _n -alkyl, SO ₂ —NH-alkyl, NH-alkyl, N(alkyl) ₂ or NH ₂ (wherein alkyl is preferably C _1-3 -alkyl).

R ^8a is preferably branched C _1-5 -alkyl, preferably methyl, ethyl, n- propyl, isopropyl, n-butyl, isobutyl, 1-methylpropyl and tert-butyl .

HZ is preferably a mono- or bicyclic heterocycle which may be substituted by one or more substituents selected from halogen, C _1-10 -alkyl which may be optionally substituted by halogen, C _6-10 -aryl or C _6-10 -aralkyl.

HZ is particularly preferably a substituted pyrrole, pyrazole, imidazole, quinazoline or dihydroquinazoline, which is substituted in the ortho position (relative to the substituent containing R1a etc.) by an optionally substituted benzyl group. In addition, the substituted pyrrole, pyrazole, imidazole or quinazoline may preferably be substituted by an oxo group (in the case of dihydroquinazoline) or by a phenyl group substituted by 1 or 2 halogen atoms. HZ is particularly preferably a substituted pyrrole.

A preferred KSP inhibitor for use is Ispins. Another preferred KSP inhibitor is Arry-520.

Other particularly preferred compounds have the following formula (IIa) or (II):

Formula (IIa), and salts, solvates and salts of solvates thereof:

in

_X1 represents N, _X2 represents N and _X3 represents C; or

_X1 represents N, _X2 represents C and _X3 represents N; or

_X1 represents CH or CF, _X2 represents C and _X3 represents N; or

_X1 represents NH, _X2 represents C and _X3 represents C; or

_X1 represents CH or CF, _X2 represents N and _X3 represents C;

(wherein X ₁ represents CH, X ₂ represents C and X ₃ represents N is preferred);

R ¹ represents H, –L-#1, –MOD or -(CH ₂ ) _0-3 Z, wherein Z represents -H, -NHY ³ , -OY ³ , -SY ³ , halogen, -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ , -(CH ₂ CH ₂ O) _0-3 -(CH ₂ ) _0-3 Z' (e.g., -(CH ₂ ) _0-3 Z') or -CH(CH ₂ W)Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, NH ₂ , SO ₃ H, COOH, -NH-CO-CH ₂ -CH ₂ -CH(NH ₂ )COOH or -(CO-NH-CHY ⁴ ) _1-3 COOH, wherein W represents H or OH,

wherein Y ⁴ independently of one another represent linear or branched C _1-6 -alkyl optionally substituted by -NHCONH ₂ , or represent aryl or benzyl optionally substituted by -NH ₂ ;

R ² represents –L-#1, H, -MOD, -CO-CHY ⁴ -NHY ⁵ or -(CH ₂ ) _0-3 Z,

wherein Z represents -H, halogen, -OY ³ , -SY ³ , NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH;

wherein Y ⁴ independently of one another represents linear or branched C _1-6 alkyl optionally substituted by -NHCONH ₂ , or represents aryl or benzyl optionally substituted by -NH ₂ , and Y ⁵ represents H or -CO-CHY ⁶ -NH ₂ , wherein Y ⁶ represents linear or branched C _1-6 -alkyl;

R ⁴ represents –L-#1, H, -CO-CHY ⁴ -NHY ⁵ or -(CH ₂ ) _0-3 Z,

wherein Z represents -H, halogen, -OY ³ , -SY ³ , NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH;

wherein Y ⁴ independently of one another represents linear or branched C _1-6 alkyl optionally substituted by -NHCONH ₂ , or represents aryl or benzyl optionally substituted by -NH ₂ , and Y ⁵ represents H or -CO-CHY ⁶ -NH ₂ , wherein Y ⁶ represents linear or branched C _1-6 -alkyl;

or R ² and R ⁴ together (forming a pyrrolidine ring) represent –CH ₂ -CHR ¹⁰ - or –CHR ¹⁰ -CH ₂ -, wherein R ¹⁰ represents L-#1, H, NH ₂ , SO ₃ H, COOH, SH or OH;

A represents CO, SO, SO ₂ , SO ₂ NH or CNNH;

R ³ represents –L-#1, –MOD or an optionally substituted alkyl, cycloalkyl, aryl, heteroaryl, heteroalkyl, heterocycloalkyl, preferably C _1-10 -alkyl, C _6-10 -aryl or C _6-10 -aralkyl, C 5-10 -heteroalkyl, C _1-10 -alkyl-OC _6-10 -aryl or C _5-10 -heterocycloalkyl, which may be substituted by 1-3 –OH groups, _1-3 halogen atoms, 1-3 haloalkyl groups (each having 1-3 halogen atoms), 1-3 O-alkyl groups, 1-3 –SH groups, 1-3 -S-alkyl groups, 1-3 -O-CO-alkyl groups, 1-3 -O-CO-NH-alkyl groups, 1-3 -NH-CO-alkyl groups, 1-3 -NH-CO-NH-alkyl groups, 1-3 -S(O) _n -alkyl groups, 1-3 -SO ₂ -NH-alkyl, 1-3 -NH-alkyl, 1-3 -N(alkyl) ₂ groups, 1-3 -NH((CH ₂ CH ₂ O) _1-20 H) groups, 1-3 -NH ₂ groups or 1-3 -(CH ₂ ) _0-3 Z groups, wherein Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ , wherein Y ¹ and Y ² independently of each other represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H, -(CH ₂ ) _0-3 -CH(NHCOCH ₃ )Z', -(CH ₂ ) _0-3 -CH(NH ₂ )Z' or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH (wherein "alkyl" is preferably C _1-10 -alkyl);

R ⁵ represents –L-#1, H, -MOD, NH ₂ , NO ₂ , halogen (especially F, Cl, Br), -CN, CF ₃ , -OCF ₃ , -CH ₂ F, -CH ₂ F, SH or -(CH ₂ ) _0-3 Z, wherein Z represents -H, -OY ³ , -SY ³ , halogen, NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH;

^R6 and ^R7 independently of one another represent H, cyano, (optionally fluorinated) _C1-10 -alkyl, (optionally fluorinated) _C2-10 -alkenyl, (optionally fluorinated) _C2-10 -alkynyl, hydroxy, _NO2 , _NH2 , COOH or halogen (especially F, Cl, Br),

R ⁸ represents (optionally fluorinated) C _1-10 -alkyl, (optionally fluorinated) C _2-10 -alkenyl, (optionally fluorinated) C _2-10 -alkynyl, (optionally fluorinated) C _4-10 -cycloalkyl or –(CH ₂ ) _0-2 -(HZ ² ), wherein HZ ² represents a 4- to 7-membered heterocycle (preferably oxetane) having up to 2 heteroatoms selected from N, O and S, wherein each of these groups may be substituted by –OH, CO ₂ H or NH ₂ or L-#1;

wherein one or none of the substituents R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁸ and R ¹⁰ represents (or contains in the case of R ⁸ ) -L-#1,

L represents a linker and #1 represents a bond to the conjugate or a derivative thereof,

R ⁹ represents H, F, CH ₃ , CF ₃ , CH ₂ F or CHF ₂ ;

Where –MOD stands for –(NR ¹⁰ ) _n -(G1) _o -G2-H, where

R ¹⁰ represents H or C ₁ -C ₃ -alkyl;

G1 represents –NHCO-, -CONH- or (wherein, if G1 represents –NHCO- or , R ¹⁰ does not represent NH ₂ );

n is 0 or 1;

o is 0 or 1; and

G2 represents a straight-chain and/or branched hydrocarbon radical having 1 to 10 carbon atoms and may be interrupted one or more times by one or more of the groups -O-, -S-, -SO-, SO ₂ , -NR ^y -, -NR ^y CO-, CONR ^y -, -NR ^y NR ^y -, -SO ₂ NR ^y NR ^y -, -CONR ^y NR ^y - (wherein R ^y represents H, phenyl, C ₁ -C ₁₀ -alkyl, C ₂ -C ₁₀ -alkenyl or C ₂ -C ₁₀ -alkynyl, each of which may be substituted by -NHCONH ₂ , -COOH, -OH, -NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid), -CO-, -CR ^x =NO- (wherein R x represents H, C ₁ -C ₃ -alkyl or phenyl), wherein the hydrocarbon chain, including side chains, if present, may be interrupted one or more times by -NHCONH ₂ , —COOH, —OH, —NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid, wherein the group —MOD preferably has at least one group —COOH.

Formula (II), and salts, solvates and salts of solvates thereof:

in

_X1 represents N, _X2 represents N and _X3 represents C; or

_X1 represents N, _X2 represents C and _X3 represents N; or

_X1 represents CH or CF, _X2 represents C and _X3 represents N; or

_X1 represents NH, _X2 represents C and _X3 represents C; or

_X1 represents CH, _X2 represents N and _X3 represents C

R ¹ represents H, -L-#1 or -(CH ₂ ) _0-3 Z, wherein Z represents -H, -NHY ³ , -OY ³ , -SY ³ , halogen, -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently of one another represent H, NH ₂ , -(CH ₂ CH ₂ O) _0-3 -(CH ₂ ) _0-3 Z' (e.g. -(CH ₂ ) _0-3 Z') or -CH(CH ₂ W)Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, NH ₂ , SO ₃ H, COOH, -NH-CO-CH ₂ -CH ₂ -CH(NH ₂ )COOH or -(CO-NH-CHY ⁴ ) _1-3 COOH, wherein W represents H or OH;

wherein Y ⁴ independently of one another represent linear or branched C _1-6 -alkyl optionally substituted by -NHCONH ₂ , or represent aryl or benzyl optionally substituted by -NH ₂ ;

R ² and R ⁴ independently represent H, -L-#1, -CO-CHY ⁴ -NHY ⁵ or -(CH ₂ ) _0-3 Z, or R ² and R ⁴ together (forming a pyrrolidine ring) represent -CH ₂ -CHR ¹⁰ - or -CHR ¹⁰ -CH ₂ -, wherein R ¹⁰ represents H, -L-#1, NH ₂ , SO ₃ H, COOH, SH or OH,

wherein Z represents -H, halogen, -OY ³ , -SY ³ , NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH;

wherein Y ⁴ independently of one another represent linear or branched C _1-6 -alkyl optionally substituted by –NHCONH ₂ or represent aryl or benzyl optionally substituted by –NH ₂ , wherein Y ⁴ independently of one another represent linear or branched C _1-6 -alkyl optionally substituted by –NHCONH ₂ or represent aryl or benzyl optionally substituted by –NH ₂ , and Y ⁵ represents H or –CO—CHY ⁶ —NH ₂ , wherein Y ⁶ represents linear or branched C _1-6 -alkyl;

A represents CO, SO, SO ₂ , SO ₂ NH or CNNH;

R ³ represents an optionally substituted alkyl, aryl, heteroaryl, heteroalkyl, heterocycloalkyl, preferably -L-#1 or C _1-10 -alkyl, C _6-10 -aryl or C _6-10 -arylalkyl, C _5-10 -heteroalkyl, C _1-10 -alkyl-OC _6-10 -aryl or C _5-10 -heterocycloalkyl, which may be substituted by 1-3 -OH groups, 1-3 halogen atoms, 1-3 haloalkyl groups (each having 1-3 halogen atoms), 1-3 O-alkyl groups, 1-3 -SH groups, 1-3 -S-alkyl groups, 1-3 -O-CO-alkyl groups, 1-3 -O-CO-NH-alkyl groups, 1-3 -NH-CO-alkyl groups, 1-3 -NH-CO-NH-alkyl groups, 1-3 -S(O) _n -alkyl groups, 1-3 -SO ₂ -NH-alkyl, 1-3 -NH-alkyl, 1-3 -N(alkyl) ₂ groups, 1-3 -NH ₂ groups or 1-3 -(CH ₂ ) _0-3 Z groups, where Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ , where Y ¹ and Y ² independently of one another represent H, NH ₂ or -(CH ₂ ) _0-3 Z ', and Y ³ represents H, -(CH ₂ ) _0-3 -CH(NHCOCH ₃ )Z ', -(CH ₂ ) _0-3 -CH(NH ₂ )Z ' or -(CH ₂ ) _0-3 Z ', where Z ' represents H, SO ₃ H, NH ₂ or COOH (wherein "alkyl" preferably represents C _1-10 -alkyl);

R ⁵ represents H, F, NH ₂ , NO ₂ , halogen, SH or -(CH ₂ ) _0-3 Z, wherein Z represents -H, halogen, -OY ³ , -SY ³ , NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH;

^R6 and ^R7 independently of one another represent H, cyano, (optionally fluorinated) _C1-10 -alkyl, (optionally fluorinated) _C2-10 -alkenyl, (optionally fluorinated) _C2-10 -alkynyl, hydroxy or halogen,

R ⁸ represents (optionally fluorinated) C _1-10 -alkyl, (optionally fluorinated) C _4-10 -cycloalkyl or optionally substituted oxetane; and

R ⁹ represents H, F, CH ₃ , CF ₃ , CH ₂ F or CHF ₂ .

Compounds of formula (IIa) or (II) in which none of the substituents R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁸ and R ¹⁰ represent -L-# ¹ can be attached to a linker by replacing a hydrogen atom at R ¹ , ^{R 2} , R ³ , R ⁴ , R ⁵ or R ⁸ or at the pyrrolidine ring (R ^{10 )} formed by R 2 and R 4 in a manner known to those skilled in the art. This results in a conjugate of formula (IIa) or (II) in which one of the substituents R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁸ or R ¹⁰ represents -L-#1, L represents a linker and #1 represents a bond to the conjugate or a derivative thereof. If the KSP inhibitor according to Formula (IIa) or (II) is conjugated to a binding entity, one of the substituents R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁸ , or R ¹⁰ represents -L-#1, where L represents a linker and #1 represents a bond to the binding entity or a derivative thereof. That is, in the case of a conjugate, one of the substituents R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁸ , and R ¹⁰ represents -L-#1, where -L-#1 is attached to the binding entity, such as an antibody. Particularly preferably, one of the substituents R ¹ and R ³ represents -L-#1. The binding entity is preferably a human, humanized, or chimeric monoclonal antibody or antigen-binding fragment thereof, in particular an anti-TWEAKR antibody or antigen-binding fragment thereof or an anti-EGFR antibody or antigen-binding fragment thereof. Particularly preferred are anti-TWEAKR antibodies that specifically bind to amino acid D in position 47 (D47) of TWEAKR (SEQ ID NO: 169), in particular the anti-TWEAKR antibody TPP-2090 or the anti-EGFR antibodies cetuximab or nimotuzumab.

The group –L-#3 may also be present in the compound in place of –L-#1, where L represents a linker and #3 represents a reactive group for binding to the conjugate or a derivative thereof. Compounds containing –L-#3 are reactive compounds that react with the conjugate or a derivative thereof. #3 is preferably a group that reacts with an amino or thiol group to form a covalent bond, preferably with a cysteine residue in a protein. The cysteine residue in the protein may be naturally present in the protein, introduced biochemically, or preferably generated by prior reduction of the disulfide of the conjugate.

Particularly preferred are compounds of formula (IIa) or (II) below, wherein one of the substituents R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ¹⁰ represents —L-#1 and wherein

^X1 represents N, _X2 represents N and _X3 represents C;

_X1 represents CH or CF, _X2 represents C and _X3 represents N;

_X1 represents NH, _X2 represents C and _X3 represents C; or

_X1 represents CH, _X2 represents N and _X3 represents C,

In particular, those in which _X1 represents N, _X2 represents N and _X3 represents C; or _X1 represents CH, _X2 represents C and _X3 represents N. Particularly preferred are compounds in which _X1 represents CH, _X2 represents C and _X3 represents N.

As A, CO (carbonyl) is preferred.

R ¹ is preferably —L-#1, H, —COOH, —CONHNH ₂ , —(CH ₂ ) _1-3 NH ₂ , —CONZ″(CH ₂ ) _1-3 NH ₂ and —CONZ″CH ₂ COOH, wherein Z″ represents H or NH ₂ .

R ² and R ⁴ are preferably H, -L-#1, or R ² and R ⁴ together (forming a pyrrolidine ring) represent CH ₂ -CHR ¹⁰ - or -CHR ¹⁰ -CH ₂ -, wherein R ¹⁰ represents H or -L-#1.

R ³ is preferably –L-#1 or C _1-10 -alkyl-, which may be optionally substituted by –OH, O-alkyl, SH, S-alkyl, O-CO-alkyl, O-CO-NH-alkyl, NH-CO-alkyl, NH-CO-NH-alkyl, S(O) _n -alkyl, SO ₂ —NH-alkyl, NH-alkyl, N(alkyl) ₂ or NH ₂ (wherein alkyl is preferably C _1-3 -alkyl).

R ⁵ is preferably —L-#1, H or F.

^R6 and ^R7 are preferably independently of one another H, (optionally fluorinated) _C1-3 -alkyl, (optionally fluorinated) _C2-4 -alkenyl, (optionally fluorinated) _C2-4 -alkynyl, hydroxy or halogen,

R ⁸ is preferably a branched C _1-5 -alkyl group, in particular a group of the formula -C(CH ₃ ) ₂ -(CH ₂ ) _0-2 -R _y , wherein R _y represents -H, -OH, CO ₂ H, NH ₂ or -L-#1. Particularly preferred is a group of the formula -C(CH ₃ ) ₂ -(CH ₂ ) -R _y , wherein R _y represents -H or -L-#1.

^R9 is preferably H or F.

Particularly preferred are compounds of formula (IIa) or (II) below, wherein none or one of the substituents R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁸ and R ¹⁰ represents -L-#1, and

in

_X1 represents N, _X2 represents N and _X3 represents C;

_X1 represents CH or CF, _X2 represents C and _X3 represents N;

_X1 represents NH, _X2 represents C and _X3 represents C; or

_X1 represents CH, _X2 represents N and _X3 represents C

A represents CO (carbonyl);

R ¹ represents H, -COOH, -CONHNH ₂ , -(CH ₂ ) _1-3 NH ₂ , -CONZ''(CH ₂ ) _1-3 NH ₂ and -CONZ''CH ₂ COOH, wherein Z'' represents H or NH ₂ ;

R ² and R ⁴ represent H or R ² and R ⁴ together (forming a pyrrolidine ring) represent -CH ₂ -CHR ¹⁰ - or -CHR ¹⁰ -CH ₂ -, wherein R ¹⁰ represents H or -L-#1;

R ³ represents phenyl which may be mono- or polysubstituted by halogen (in particular F) or optionally fluorinated C _1-3 -alkyl, or represents optionally fluorinated C 1-10 -alkyl which may be optionally substituted by —OY ⁴ , —SY ⁴ , —O-CO-Y ⁴ , —O-CO-NH-Y ⁴ , NH-CO-Y ⁴ , —NH-CO-NH- ^{Y 4} , S(O) _n -Y ⁴ (wherein n represents 0, 1 or 2), —SO ₂ —NH-Y ⁴ , NH _- Y ⁴ or N(Y ⁴ ) ₂ , where Y ⁴ represents H, phenyl (optionally mono- or polysubstituted by halogen (in particular F) or optionally fluorinated C _1-3 -alkyl) or alkyl (wherein the alkyl may be substituted by —OH, —COOH and/or —NHCO-C _1-3 -alkyl, and where alkyl preferably represents C _1-3 -alkyl);

R ³ is particularly preferably substituted by -OH, O-alkyl, SH, S-alkyl, O-CO-alkyl, O-CO-NH-alkyl, NH-CO-alkyl, NH-CO-NH-alkyl, S(O) _n -alkyl, SO ₂ -NH-alkyl, NH-alkyl, N(alkyl) ₂ or NH ₂ (wherein alkyl preferably refers to C _1-3 -alkyl)

R ⁵ represents H or F;

R ⁶ and R ⁷ independently of one another represent H, (optionally fluorinated) C _1-3 -alkyl, (optionally fluorinated) C _2-4 -alkenyl, (optionally fluorinated) C _2-4 -alkynyl, hydroxy or halogen;

R ⁸ represents a branched C _1-5 -alkyl group; and

^R9 represents H or F.

Furthermore, it is preferred that (alone or in combination)

●R ¹ represents –L-#1, COOH or H,

R ² and R ⁴ independently represent -L-#1 or H, or R ² and R ⁴ together (forming a pyrrolidine ring) represent -CH ₂ -CHR ¹⁰ - or -CHR ¹⁰ -CH ₂ -, wherein R ¹⁰ represents H or -L-#1,

●A stands for CO,

●R ³ represents -(CH ₂ )OH, -CH(CH ₃ )OH, -CH ₂ SCH ₂ CH(COOH)NHCOCH ₃ , -CH(CH ₃ )OCH ₃ , phenyl which may be substituted by 1-3 halogen atoms, 1-3 amino groups or 1-3 alkyl groups (which may be optionally halogenated), or represents –L-#1,

●R ⁵ represents –L-#1 or H,

R ⁶ and R ⁷ independently of one another represent H, C _1-3 -alkyl or halogen; R ⁶ and R ⁷ in particular represent F;

● R ⁸ represents C _1-4 -alkyl (preferably tert-butyl); and/or

●R ⁹ represents H,

• wherein one of the substituents R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ¹⁰ represents -L-#1.

Furthermore, according to the present invention, it is preferred that

●R ¹ represents –L-#1, COOH or H,

R ² and R ⁴ independently represent -L-#1 or H, or R ² and R ⁴ together (forming a pyrrolidine ring) represent -CH ₂ -CHR ¹⁰ - or -CHR ¹⁰ -CH ₂ -, wherein R ¹⁰ represents H or -L-#1,

●A stands for CO,

●R ³ represents -(CH ₂ )OH, -CH(CH ₃ )OH, -CH ₂ SCH ₂ CH(COOH)NHCOCH ₃ , -CH(CH ₃ )OCH ₃ , phenyl which may be substituted by 1-3 halogen atoms, 1-3 amino groups or 1-3 alkyl groups (which may be optionally halogenated), or represents –L-#1,

●R ⁵ represents –L-#1 or H,

R ⁶ and R ⁷ independently of one another represent H, C _1-3 -alkyl or halogen; R ⁶ and R ⁷ in particular represent F;

● R ⁸ represents C _1-4 -alkyl (preferably tert-butyl); and

●R ⁹ represents H,

• wherein one of the substituents R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ¹⁰ represents -L-#1.

Other particularly preferred compounds have the following formula (IIIa) or (III) and their salts, solvates and salts of solvates:

Formula (IIIa):

in

_X1 represents N, _X2 represents N and _X3 represents C; or

_X1 represents N, _X2 represents C and _X3 represents N; or

_X1 represents CH or CF, _X2 represents C and _X3 represents N; or

_X1 represents NH, _X2 represents C and _X3 represents C; or

_X1 represents CH, _X2 represents N and _X3 represents C

(wherein X ₁ represents CH, X ₂ represents C and X ₃ represents N is preferred);

R ¹ represents H, –L-BINDER, –MOD or –(CH ₂ ) _0-3 Z, wherein Z represents –H, –NHY ³ , –OY ³ , –SY ³ , halogen, –CO-NY ¹ Y ² or –CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ , -(CH ₂ CH ₂ O) _0-3 -(CH ₂ ) _0-3 Z' (e.g., -(CH ₂ ) _0-3 Z') or -CH(CH ₂ W)Z', and Y ³ represents H or -(CH ₃ ) _0-3 Z', wherein Z' represents H, NH ₂ , SO ₃ H, -COOH, -NH-CO-CH ₂ -CH ₂ -CH(NH ₂ )COOH or -(CO-NH-CHY ⁴ ) _1-3 COOH, wherein W represents H or OH,

wherein Y ⁴ independently of one another represent linear or branched C _1-6 -alkyl optionally substituted by -NHCONH ₂ , or represent aryl or benzyl optionally substituted by -NH ₂ ;

R ² represents H, -L-BINDER, -MOD, -CO-CHY ⁴ -NHY ⁵ or -(CH ₂ ) _0-3 Z, wherein Y ⁴ independently of one another represent linear or branched C _1-6 alkyl optionally substituted by -NHCONH ₂ , or represent aryl or benzyl optionally substituted by -NH ₂ , and Y ⁵ represents H or -CO-CHY ⁶ -NH ₂ , wherein Y ⁶ represents linear or branched C _1-6 -alkyl;

wherein Z represents -H, halogen, -OY ³ , -SY ³ , NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH;

R ⁴ represents H, -L-BINDER, -CO-CHY ⁴ -NHY ⁵ or -(CH ₂ ) _0-3 Z,

wherein Z represents -H, halogen, -OY ³ , -SY ³ , NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH;

wherein Y ⁴ independently of one another represents linear or branched C _1-6 alkyl optionally substituted by -NHCONH ₂ , or represents aryl or benzyl optionally substituted by -NH ₂ , and Y ⁵ represents H or -CO-CHY ⁶ -NH ₂ , wherein Y ⁶ represents linear or branched C _1-6 -alkyl;

or R ² and R ⁴ together (forming a pyrrolidine ring) represent –CH ₂ -CHR ¹⁰ - or –CHR ¹⁰ -CH ₂ -, wherein R ¹⁰ represents –L-BINDER, H, NH ₂ , SO ₃ H, COOH, SH or OH;

A represents CO, SO, SO ₂ , SO ₂ NH or CNNH;

R ³ represents -L-BINDER, -MOD or optionally substituted alkyl, cycloalkyl, aryl, heteroaryl, heteroalkyl, heterocycloalkyl, preferably -L-#1 or C _1-10 -alkyl, C _6-10 -aryl or C _6-10 -arylalkyl, C 5-10 -heteroalkyl, C _1-10 -alkyl-OC _6-10 -aryl or C _5-10 -heterocycloalkyl, which may be substituted by 1-3 -OH groups, _1-3 halogen atoms, 1-3 haloalkyl groups (each having 1-3 halogen atoms), 1-3 O-alkyl groups, 1-3 -SH groups, 1-3 -S-alkyl groups, 1-3 -O-CO-alkyl groups, 1-3 -O-CO-NH-alkyl groups, 1-3 -NH-CO-alkyl groups, 1-3 -NH-CO-NH-alkyl groups, 1-3 -S(O) _n -alkyl groups, 1-3 -SO ₂ -NH-alkyl, 1-3 -NH-alkyl, 1-3 -N(alkyl) ₂ groups, 1-3 -NH ₂ groups or 1-3 -(CH ₂ ) _0-3 Z groups, where Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ , where Y ¹ and Y ² independently of one another represent H, NH ₂ or -(CH ₂ ) _0-3 Z ', and Y ³ represents H, -(CH ₂ ) _0-3 -CH(NHCOCH ₃ )Z ', -(CH ₂ ) _0-3 -CH(NH ₂ )Z ' or -(CH ₂ ) _0-3 Z ', where Z ' represents H, SO ₃ H, NH ₂ or COOH (wherein "alkyl" preferably represents C _1-10 -alkyl);

R ⁵ represents -L-BINDER, H, NH ₂ , NO ₂ , halogen (especially F, Cl, Br), -CN, CF ₃ , -OCF ₃ , -CH ₂ F, -CH ₂ F, SH or -(CH ₂ ) _0-3 Z, wherein Z represents -H, -OY ³ , -SY ³ , halogen, NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH;

^R6 and ^R7 independently of one another represent H, cyano, (optionally fluorinated) _C1-10 -alkyl, (optionally fluorinated) _C2-10 -alkenyl, (optionally fluorinated) _C2-10 -alkynyl, hydroxy, _NO2 , _NH2 , COOH or halogen (especially F, Cl, Br),

R ⁸ represents (optionally fluorinated) C _1-10 -alkyl, (optionally fluorinated) C _2-10 -alkenyl, (optionally fluorinated) C _2-10 -alkynyl, (optionally fluorinated) C _4-10 -cycloalkyl or –(CH ₂ ) _0-2 -(HZ ² ), wherein HZ ² represents a 4- to 7-membered heterocycle having up to 2 heteroatoms selected from N, O and S, wherein each of these groups may be substituted by –OH, CO ₂ H or NH ₂ or –L-BINDER;

R ⁹ represents H, F, CH ₃ , CF ₃ , CH ₂ F or CHF ₂ ;

Where –MOD stands for –(NR ¹⁰ ) _n -(G1) _o -G2-H, where

R ¹⁰ represents H or C ₁ -C ₃ -alkyl;

G1 represents –NHCO-, -CONH- or (wherein, if G1 represents –NHCO- or , R ¹⁰ does not represent NH ₂ );

n is 0 or 1;

o is 0 or 1; and

G2 represents a straight-chain and/or branched hydrocarbon radical having 1 to 10 carbon atoms and may be interrupted one or more times by one or more of the groups -O-, -S-, -SO-, SO ₂ , -NR ^y -, -NR ^y CO-, CONR ^y -, -NR ^y NR ^y -, -SO ₂ NR ^y NR ^y -, -CONR ^y NR ^y - (wherein R ^y represents H, phenyl, C ₁ -C ₁₀ -alkyl, C ₂ -C ₁₀ -alkenyl or C ₂ -C ₁₀ -alkynyl, each of which may be substituted by -NHCONH ₂ , -COOH, -OH, -NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid), -CO-, -CR ^x =NO- (wherein R x represents H, C ₁ -C ₃ -alkyl or phenyl), wherein the hydrocarbon chain, including side chains, if present, may be interrupted one or more times by -NHCONH ₂ , —COOH, —OH, —NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid, wherein the group —MOD preferably has at least one group —COOH.

In the case of binder conjugates of KSP inhibitors of the formula (IIIa), at most one representative of R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁸ and R ¹⁰ (instead of one of the conditions given above) may represent -L-BINDER, where L represents a linker and BINDER represents a binder or a derivative thereof, where the binder can optionally be attached to a plurality of active substance molecules.

Formula (III) and its salts, solvates and salts of solvates:

in

_X1 represents N, _X2 represents N and _X3 represents C, or _X1 represents CH, _X2 represents C and _X3 represents N, or _X1 represents NH, _X2 represents C and _X3 represents C, or _X1 represents CH, _X2 represents N and _X3 represents C;

R1 represents -L-BINDER, H or -(CH ₂ ) _0-3 Z, wherein Z represents -H, -NHY ³ , -OY ³ , -SY ³ , halogen, -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ , -(CH ₂ CH ₂ O) _0-3 -(CH ₂ ) _0-3 Z' or -CH(CH ₂ W)Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, NH ₂ , SO ₃ H, COOH, -NH-CO-CH ₂ -CH ₂ -CH(NH ₂ )COOH or -(CO-NH-CHY ⁴ ) _1-3 COOH; wherein W represents H or OH;

wherein Y ⁴ independently of one another represent linear or branched C _1-6 -alkyl optionally substituted by -NHCONH ₂ , or represent aryl or benzyl optionally substituted by -NH ₂ ;

R ² and R ⁴ independently represent -L-BINDER, H, -CO-CHY ⁴ -NHY ⁵ or -(CH ₂ ) _0-3 Z, or R ² and R ⁴ together (forming a pyrrolidine ring) represent -CH ₂ -CHR ¹⁰ - or -CHR ¹⁰ -CH ₂ -, wherein R ¹⁰ represents L-#1, H, NH ₂ , SO ₃ H, COOH, SH or OH,

wherein Z represents -H, halogen, -OY ³ , -SY ³ , NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH;

wherein Y ⁴ independently of one another represents linear or branched C _1-6 alkyl optionally substituted by -NHCONH ₂ , or represents aryl or benzyl optionally substituted by -NH ₂ , and Y ⁵ represents H or -CO-CHY ⁶ -NH ₂ , wherein Y ⁶ represents linear or branched C _1-6 -alkyl;

A represents CO, SO, SO ₂ , SO ₂ NH or CNNH;

R ³ represents –L-BINDER or optionally substituted alkyl, aryl, heteroaryl, heteroalkyl, heterocycloalkyl, preferably –L-#1 or C _1-10 -alkyl, C _6-10 -aryl or C _{6-10 -arylalkyl, C 5-10} -heteroalkyl, C _1-10 -alkyl-OC _6-10 -aryl or C _5-10 -heterocycloalkyl, which may be substituted by 1-3 –OH groups, 1-3 halogen atoms, _1-3 haloalkyl groups (each having 1-3 halogen atoms), 1-3 O-alkyl groups, 1-3 –SH groups, 1-3 –S-alkyl groups, 1-3 –O-CO-alkyl groups, 1-3 –O-CO-NH-alkyl groups, 1-3 –NH-CO-alkyl groups, 1-3 –NH-CO-NH-alkyl groups, 1-3 –S(O) _n -alkyl groups, 1-3 –SO ₂ -NH-alkyl, 1-3 -NH-alkyl, 1-3 -N(alkyl) ₂ groups, 1-3 -NH ₂ groups or 1-3 -(CH ₂ ) _0-3 Z groups, where Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ , where Y ¹ and Y ² independently of one another represent H, NH ₂ or -(CH ₂ ) _0-3 Z ', and Y ³ represents H, -(CH ₂ ) _0-3 -CH(NHCOCH ₃ )Z ', -(CH ₂ ) _0-3 -CH(NH ₂ )Z ' or -(CH ₂ ) _0-3 Z ', where Z ' represents H, SO ₃ H, NH ₂ or COOH (wherein "alkyl" preferably represents C _1-10 -alkyl);

R ⁵ represents -L-BINDER, H, F, NH ₂ , NO ₂ , halogen, SH or -(CH ₂ ) _0-3 Z, wherein Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH;

wherein L represents a linker and BINDER represents a binder or a derivative thereof, wherein the binder can optionally be linked to a plurality of active substance molecules,

^R6 and ^R7 independently of one another represent H, cyano, (optionally fluorinated) _C1-10 -alkyl, (optionally fluorinated) _C2-10 -alkenyl, (optionally fluorinated) _C2-10 -alkynyl, hydroxy or halogen,

R ⁸ represents (optionally fluorinated) C _1-10 -alkyl, (optionally fluorinated) C _4-10 -cycloalkyl or optionally substituted oxetane; and

R ⁹ represents H, F, CH ₃ , CF ₃ , CH ₂ F or CHF ₂ .

Furthermore, the following KSP inhibitors and their conjugates are preferred according to the present invention:

Formula (IIIb):

wherein X ₁ , X ₂ , X ₃ have the same meanings as in formula (IIIa) or (III) (wherein preferably X ₁ represents CH, X ₂ represents C and X ₃ represents N), R ¹ , R ² , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ and R ⁹ have the same meanings as in formula (IIIa) or (III), A represents CO, B represents a single bond, –O—CH ₂ – or –CH ₂ -O— and R ²⁰ represents NH ₂ , F, CF ₃ or CH ₃ , and n represents 0, 1 or 2.

Formula (IIIc):

wherein X ₁ , X ₂ , X ₃ have the same meanings as in formula (IIIa) or (III) (wherein preferably X ₁ represents CH, X ₂ represents C and X ₃ represents N), A, R ¹ , R ³ , R ⁶ , R ⁷ , R ⁸ and R ⁹ have the same meanings as in formula (IIIa) or (III), A preferably represents CO and R ³ represents —CH ₂ OH, —CH ₂ OCH ₃ , CH(CH ₃ )OH or CH(CH ₃ )OCH ₃ .

Formula (IIId):

wherein X ₁ , X ₂ , X ₃ have the same meanings as in formula (IIIa) or (III) (wherein preferably X ₁ represents CH, X ₂ represents C and X ₃ represents N), A, R ³ , R ⁶ , R ⁷ , R ⁸ and R ⁹ have the same meanings as in formula (IIIa) or (III), wherein A preferably represents CO and R ³ represents —CH ₂ —S _x —(CH ₂ ) _0-4 —CHY ⁵ —COOH, wherein x is 0 or 1 and Y ⁵ represents H or NHY ⁶ , wherein Y ⁶ represents H or —COCH ₃ .

Formula (IIIe):

wherein X ₁ represents CH, X ₂ represents C and X ₃ represents N, A, R ³ , R ⁶ , R ⁷ , R ⁸ and R ⁹ have the same meanings as in formula (IIIa) or (III) and R ¹ represents -L-BINDER.

Furthermore, it is preferred that, among the compounds of formula (III), (IIIa), (IIIb), (IIIc), (IIId) and (IIIe) (alone or in combination):

●Z represents Cl or Br;

●R ¹ represents -(CH ₂ ) _0-3 Z, wherein Z represents -CO-NY ¹ Y ² , wherein Y ² represents -(CH ₂ CH ₂ O) _0-3 -(CH ₂ ) _0-3 Z′, and Y ¹ represents H, NH ₂ or -(CH ₂ CH ₂ O) _0-3 -(CH ₂ ) _0-3 Z′;

●Y ¹ represents H, Y ² represents -(CH ₂ CH ₂ O) ₃ -CH ₂ CH ₂ Z' and Z' represents -COOH;

●Y ¹ represents H, Y ² represents -CH ₂ CH ₂ Z' and Z' represents -(CONHCHY ⁴ ) ₂ COOH;

● Y ¹ represents H, Y ² represents -CH ₂ CH ₂ Z', Z' represents -(CONHCHY ⁴ ) ₂ COOH and one of Y ⁴ represents isopropyl and the other represents -(CH ₂ ) ₃ -NHCONH ₂ ;

● Y ¹ represents H, Y ² represents -CH ₂ CH ₂ Z', Z' represents -(CONHCHY ⁴ ) ₂ COOH and one of Y ⁴ represents -CH ₃ and the other represents -(CH ₂ ) ₃ -NHCONH ₂ ;

● Y ⁴ represents a linear or branched C _1-6 -alkyl group optionally substituted by -NHCONH ₂ ;

● at least one Y ⁴ represents a group selected from isopropyl and -CH ₃ ;

● Y ¹ represents H, Y ² represents -CH ₂ CH ₂ Z', Z' represents -CONHCHY ⁴ COOH and Y ⁴ represents aryl or benzyl optionally substituted by -NH ₂ ;

● ^Y4 represents aminobenzyl;

● R ² represents –(CH ₂ ) _0-3 Z and Z represents –SY ³ ;

● R ⁴ represents -CO-CHY ⁴ -NHY ⁵ and Y ⁵ represents H;

• R ⁴ represents —CO—CHY ⁴ —NHY ⁵ and Y ⁵ represents —CO—CHY ⁶ —NH ₂ ;

● Y ⁴ represents a linear or branched C _1-6 -alkyl group optionally substituted by —NHCONH ₂ .

Furthermore, it is preferred that R ¹ , R ² or R ³ in formula (IIa) or (IIIa) represents -MOD, and in particular R ⁴ represents -L-#1 or -L-BINDER (in particular, -L is a cleavable linker that is directly cleaved at -NR ⁴ or -N—L-#1 or -L-BINDER to replace R ⁴ or L with H).

Particularly preferably, R ³ represents -MOD and R ¹ or R ⁴ represents -L-#1 or -L-BINDER,

Where –MOD stands for –(NR ¹⁰ ) _n -(G1) _o -G2-H, where

R ¹⁰ represents H or C ₁ -C ₃ -alkyl;

G1 represents –NHCO-, -CONH- or (wherein, if G1 represents –NHCO- or , R ¹⁰ does not represent NH ₂ );

n is 0 or 1;

o is 0 or 1; and

G2 represents a straight-chain and/or branched hydrocarbon radical having 1 to 10 carbon atoms and may be interrupted one or more times by one or more of the groups -O-, -S-, -SO-, SO ₂ , -NR ^y -, -NR ^y CO-, CONR ^y -, -NR ^y NR ^y -, -SO ₂ NR ^y NR ^y -, -CONR ^y NR ^y - (wherein R ^y represents H, phenyl, C ₁ -C ₁₀ -alkyl, C ₂ -C ₁₀ -alkenyl or C ₂ -C ₁₀ -alkynyl, each of which may be substituted by -NHCONH ₂ , -COOH, -OH, -NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid), -CO-, -CR ^x =NO- (wherein R x represents H, C ₁ -C ₃ -alkyl or phenyl), wherein the hydrocarbon chain, including side chains, if present, may be interrupted one or more times by -NHCONH ₂ , -COOH, -OH, -NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid substitution, wherein the group -MOD preferably has at least one group -COOH;

Particularly preferably, the group -MOD has a (preferably terminal) -COOH group, for example in a betaine group. The group -MOD preferably has the formula _-CH2 - _Sx- ( _CH2 ) _0-4- ^CHY5 -COOH, where x is 0 or 1 and ^Y5 represents H or ^NHY6 , where ^Y6 represents H or _-COCH3 .

Other particularly preferred compounds have the following formula (IV) and their salts, solvates and salts of solvates:

in

_X1 represents N, _X2 represents C and _X3 represents N;

R ¹ represents –L-BINDER, H or -(CH ₂ ) _0-3 Z, wherein Z represents -H, -NHY ³ , -OY ³ , -SY ³ , halogen, -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently of one another represent H, NH ₂ , -(CH ₂ CH ₂ O) _0-3 -(CH ₂ ) _0-3 Z' or -CH(CH ₂ W)Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, NH ₂ , SO ₃ H, COOH, -NH-CO-CH ₂ -CH ₂ -CH(NH ₂ )COOH or -(CO-NH-CHY ⁴ ) _1-3 COOH;

wherein Y ⁴ independently of one another represent a linear or branched C _1-6 -alkyl group optionally substituted by -NHCONH ₂ , or represent an aryl group or a benzyl group optionally substituted by -NH ₂ ;

R ² and R ⁴ independently represent -L-BINDER, H, -CO-CHY ⁴ -NHY ⁵ or -(CH ₂ ) _0-3 Z, or R ² and R ⁴ together (forming a pyrrolidine ring) represent -CH ₂ -CHR ¹⁰ - or -CHR ¹⁰ -CH ₂ -, wherein R ¹⁰ represents L-#1, H, NH ₂ , SO ₃ H, COOH, SH or OH,

wherein Z represents -H, halogen, -OY ³ , -SY ³ , NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH;

wherein Y ⁴ independently of one another represents linear or branched C _1-6 alkyl optionally substituted by -NHCONH ₂ , or represents aryl or benzyl optionally substituted by -NH ₂ , and Y ⁵ represents H or -CO-CHY ⁶ -NH ₂ , wherein Y ⁶ represents linear or branched C _1-6 -alkyl;

A represents CO, SO, SO ₂ , SO ₂ NH or CNNH;

R ³ represents -L-BINDER, optionally substituted alkyl, aryl, heteroaryl, heteroalkyl, heterocycloalkyl or -CH ₂ -S _x -(CH ₂ ) _0-4 -CHY ⁵ -COOH, wherein x is 0 or 1, and Y ⁵ represents H or NHY ⁶ , wherein Y ⁶ represents H or -COCH ₃ , preferably -L-BINDER or C _1-10 -alkyl, C _6-10 -aryl or C _6-10 -aralkyl, C _5-10 -heteroalkyl, C _1-10 -alkyl-OC _6-10 -aryl or C _5-10 -heterocycloalkyl, which may be substituted by 1-3 -OH groups, 1-3 halogen atoms, 1-3 haloalkyl groups (each having 1-3 halogen atoms), 1-3 O-alkyl groups, 1-3 -SH groups, 1-3 -S-alkyl groups, 1-3 -O-CO-alkyl groups, 1-3 -O-CO-NH-alkyl groups, 1-3 -NH-CO-alkyl groups, 1-3 -NH-CO-NH-alkyl groups, 1-3 -S(O) _n -alkyl groups, 1-3 -SO ₂ -NH-alkyl groups, 1-3 -NH-alkyl groups, 1-3 -N(alkyl) ₂ groups, 1-3 -NH ₂ groups or 1-3 -(CH ₂ ) _0-3 Z groups, where Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ , where Y ¹ and Y ² independently of one another represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H, -(CH ₂ ) _0-3 -CH(NHCOCH ₃ )Z', -(CH ₂ ) _0-3 -CH(NH ₂ )Z' or -(CH ₂ ) _0-3 Z', where Z' represents H, SO ₃ H, NH ₂ or COOH (wherein "alkyl" preferably represents C _1-10 -alkyl);

R ⁵ represents -L-BINDER, H, F, NH ₂ , NO ₂ , halogen, SH or -(CH ₂ ) _0-3 Z, wherein Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH;

wherein L represents a linker and BINDER represents a binder or a derivative thereof, wherein the binder can optionally be linked to a plurality of active substance molecules,

^R6 and ^R7 independently of one another represent H, cyano, (optionally fluorinated) _C1-10 -alkyl, (optionally fluorinated) _C2-10 -alkenyl, (optionally fluorinated) _C2-10 -alkynyl, hydroxy or halogen,

R ⁸ represents (optionally fluorinated) C _1-10 -alkyl, (optionally fluorinated) C _4-10 -cycloalkyl or optionally substituted oxetane; and

R ⁹ represents H, F, CH ₃ , CF ₃ , CH ₂ F or CHF ₂ ;

Provided that R ¹ , R ² and R ⁴ do not represent H at the same time.

Furthermore, it is preferred that (alone or in combination) in formula (IIa), (II), (III), (IIIa), (IIIb), (IIIc), (IIId), (IIIe) or (IV):

●Z represents Cl or Br;

● R ¹ represents -(CH ₂ ) _0-3 Z, wherein Z represents -CO-NY ¹ Y ² , wherein Y ² represents -(CH ₂ CH ₂ O) _0-3 -(CH ₂ ) _0-3 Z′ and Y ¹ represents H, NH ₂ or -(CH ₂ CH ₂ O) _0-3 -(CH ₂ ) _0-3 Z′;

●Y ¹ represents H, Y ² represents -(CH ₂ CH ₂ O) ₃ -CH ₂ CH ₂ Z' and Z' represents -COOH;

●Y ¹ represents H, Y ² represents -CH ₂ CH ₂ Z' and Z' represents -(CONHCHY ⁴ ) ₂ COOH;

● Y ¹ represents H, Y ² represents -CH ₂ CH ₂ Z', Z' represents -(CONHCHY ⁴ ) ₂ COOH and one Y ⁴ represents isopropyl and the other represents -(CH ₂ ) ₃ -NHCONH ₂ ;

● Y ¹ represents H, Y ² represents -CH ₂ CH ₂ Z', Z' represents -(CONHCHY ⁴ ) ₂ COOH and one Y ⁴ represents -CH ₃ and the other represents -(CH ₂ ) ₃ -NHCONH ₂ ;

● Y ⁴ represents a linear or branched C _1-6 -alkyl group optionally substituted by -NHCONH ₂ ;

● at least one Y ⁴ represents a group selected from isopropyl and –CH ₃ ;

● Y ¹ represents H, Y ² represents -CH ₂ CH ₂ Z', Z' represents -CONHCHY ⁴ COOH and Y ⁴ represents aryl or benzyl optionally substituted by -NH ₂ ;

● ^Y4 represents aminobenzyl;

● R ² represents –(CH ₂ ) _0-3 Z and Z represents –SY ³ ;

● R ⁴ represents -CO-CHY ⁴ -NHY ⁵ and Y ⁵ represents H;

• R ⁴ represents —CO—CHY ⁴ —NHY ⁵ and Y ⁵ represents —CO—CHY ⁶ —NH ₂ ;

● Y ⁴ represents a linear or branched C _1-6 -alkyl group optionally substituted by —NHCONH ₂ .

Also preferred are compounds of formula (IIa), (II), (III), (IIIa) or (IV) and their salts, solvates and salts of solvates

in

_X1 represents N, _X2 represents N and _X3 represents C; or

_X1 represents N, _X2 represents C and _X3 represents N; or

_X1 represents CH or CF, _X2 represents C and _X3 represents N; or

_X1 represents NH, _X2 represents C and _X3 represents C; or

_X1 represents CH or CF, _X2 represents N and _X3 represents C;

(wherein X ₁ represents CH, X ₂ represents C and X ₃ represents N is preferred);

R ¹ represents H, –L-#1 or –L-BINDER, –MOD or -(CH ₂ ) _0-3 Z, wherein Z represents -H, -NHY ³ , -OY ³ , -SY ³ , halogen, -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ , -(CH ₂ CH ₂ O) _0-3 -(CH ₂ ) _0-3 Z' (e.g., -(CH ₂ ) _0-3 Z') or -CH(CH ₂ W)Z', and Y ³ represents H or -(CH ₃ ) _0-3 Z', wherein Z' represents H, NH ₂ , SO ₃ H, COOH, -NH-CO-CH ₂ -CH ₂ -CH(NH ₂ )COOH or -(CO-NH-CHY ⁴ ) _1-3 COOH, wherein W represents H or OH,

wherein Y ⁴ independently of one another represent linear or branched C _1-6 -alkyl optionally substituted by -NHCONH ₂ , or represent aryl or benzyl optionally substituted by -NH ₂ ;

R ² represents H, -CO-CHY ⁴ -NHY ⁵ or -(CH ₂ ) _0-3 Z,

wherein Z represents -H, halogen, -OY ³ , -SY ³ , NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH;

wherein Y ⁴ independently of one another represents linear or branched C _1-6 alkyl optionally substituted by -NHCONH ₂ , or represents aryl or benzyl optionally substituted by -NH ₂ , and Y ⁵ represents H or -CO-CHY ⁶ -NH ₂ , wherein Y ⁶ represents linear or branched C _1-6 -alkyl;

R ⁴ represents H;

A represents CO, SO, SO ₂ , SO ₂ NH or CNNH;

R ³ represents –L-#1 or –L-BINDER, -MOD or optionally substituted alkyl, cycloalkyl, aryl, heteroaryl, heteroalkyl, heterocycloalkyl, preferably C _1-10 -alkyl, C _6-10 -aryl or C _6-10 -arylalkyl, C _5-10 -heteroalkyl, C _1-10 -alkyl-OC _6-10 -aryl or C _5-10 -heterocycloalkyl, which may be substituted by 1-3 –OH groups, 1-3 halogen atoms, 1-3 haloalkyl groups (each having 1-3 halogen atoms), 1-3 O-alkyl groups, 1-3 –SH groups, 1-3 -S-alkyl groups, 1-3 -O-CO-alkyl groups, 1-3 -O-CO-NH-alkyl groups, 1-3 -NH-CO-alkyl groups, 1-3 -NH-CO-NH-alkyl groups, 1-3 -S(O) _n -alkyl groups, 1-3 -SO ₂ -NH-alkyl, 1-3 -NH-alkyl, 1-3 -N(alkyl) ₂ groups, 1-3 -NH((CH ₂ CH ₂ O) _1-20 H) groups, 1-3 -NH ₂ groups or 1-3 -(CH ₂ ) _0-3 Z groups, wherein Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ , wherein Y ¹ and Y ² independently of each other represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H, -(CH ₂ ) _0-3 -CH(NHCOCH ₃ )Z', -(CH ₂ ) _0-3 -CH(NH ₂ )Z' or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH (wherein "alkyl" is preferably C _1-10 -alkyl);

R ⁵ represents H, -MOD, NH ₂ , NO ₂ , halogen (especially F, Cl, Br), -CN, CF ₃ , -OCF ₃ , -CH ₂ F, -CH ₂ F, SH or -(CH ₂ ) _0-3 Z, wherein Z represents -H, -OY ³ , -SY ³ , halogen, NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH;

^R6 and ^R7 independently of one another represent H, cyano, (optionally fluorinated) _C1-10 -alkyl, (optionally fluorinated) _C2-10 -alkenyl, (optionally fluorinated) _C2-10 -alkynyl, hydroxy, _NO2 , _NH2 , COOH or halogen (especially F, Cl, Br),

R ⁸ represents (optionally fluorinated) C _1-10 -alkyl, (optionally fluorinated) C _2-10 -alkenyl, (optionally fluorinated) C _2-10 -alkynyl or (optionally fluorinated) C _4-10 -cycloalkyl;

wherein one or neither of the substituents R ¹ and R ³ represents –L-#1, –L-#1 or –L-BINDER,

L represents a linker and #1 represents a bond to a binder or a derivative thereof and BINDER represents a binder,

R ⁹ represents H, F, CH ₃ , CF ₃ , CH ₂ F or CHF ₂ ;

Where –MOD stands for –(NR ¹⁰ ) _n -(G1) _o -G2-H, where

R ¹⁰ represents H or C ₁ -C ₃ -alkyl;

G1 represents –NHCO-, -CONH- or (wherein, if G1 represents –NHCO- or , R ¹⁰ does not represent NH ₂ );

n is 0 or 1;

o is 0 or 1; and

G2 represents a straight-chain and/or branched hydrocarbon radical having 1 to 10 carbon atoms and may be interrupted one or more times by one or more of the groups -O-, -S-, -SO-, SO ₂ , -NR ^y -, -NR ^y CO-, CONR ^y -, -NR ^y NR ^y -, -SO ₂ NR ^y NR ^y -, -CONR ^y NR ^y - (wherein R ^y represents H, phenyl, C ₁ -C ₁₀ -alkyl, C ₂ -C ₁₀ -alkenyl or C ₂ -C ₁₀ -alkynyl, each of which may be substituted by -NHCONH ₂ , -COOH, -OH, -NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid), -CO-, -CR ^x =NO- (wherein R x represents H, C ₁ -C ₃ -alkyl or phenyl), wherein the hydrocarbon chain, including side chains, if present, may be interrupted one or more times by -NHCONH ₂ , —COOH, —OH, —NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid, wherein the group —MOD preferably has at least one group —COOH.

Also preferred are compounds of formula (IIa), (II), (III), (IIIa) or (IV) and their salts, solvates and salts of solvates, wherein

_X1 represents N, _X2 represents N and _X3 represents C; or

_X1 represents N, _X2 represents C and _X3 represents N; or

_X1 represents CH or CF, _X2 represents C and _X3 represents N; or

_X1 represents NH, _X2 represents C and _X3 represents C; or

_X1 represents CH or CF, _X2 represents N and _X3 represents C;

(wherein X ₁ represents CH, X ₂ represents C and X ₃ represents N is preferred);

R ¹ represents H, –L-#1 or –L-BINDER, –MOD or -(CH ₂ ) _0-3 Z, wherein Z represents -H, -NHY ³ , -OY ³ , -SY ³ , halogen, -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ , -(CH ₂ CH ₂ O) _0-3 -(CH ₂ ) _0-3 Z' (e.g., -(CH ₂ ) _0-3 Z') or -CH(CH ₂ W)Z', and Y ³ represents H or -(CH ₃ ) _0-3 Z', wherein Z' represents H, NH ₂ , SO ₃ H, COOH, -NH-CO-CH ₂ -CH ₂ -CH(NH ₂ )COOH or -(CO-NH-CHY ⁴ ) _1-3 COOH, wherein W represents H or OH,

wherein Y ⁴ independently of one another represent linear or branched C _1-6 -alkyl optionally substituted by -NHCONH ₂ , or represent aryl or benzyl optionally substituted by -NH ₂ ;

R ² represents H, -CO-CHY ⁴ -NHY ⁵ or -(CH ₂ ) _0-3 Z,

wherein Z represents -H, halogen, -OY ³ , -SY ³ , NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH;

wherein Y ⁴ independently of one another represents linear or branched C _1-6 alkyl optionally substituted by -NHCONH ₂ , or represents aryl or benzyl optionally substituted by -NH ₂ , and Y ⁵ represents H or -CO-CHY ⁶ -NH ₂ , wherein Y ⁶ represents linear or branched C _1-6 -alkyl;

R ⁴ represents H;

A represents CO, SO, SO ₂ , SO ₂ NH or CNNH;

R ³ represents –L-#1 or –L-BINDER, -MOD or optionally substituted alkyl, cycloalkyl, aryl, heteroaryl, heteroalkyl, heterocycloalkyl, preferably C _1-10 -alkyl, C _6-10 -aryl or C _6-10 -arylalkyl, C _5-10 -heteroalkyl, C _1-10 -alkyl-OC _6-10 -aryl or C _5-10 -heterocycloalkyl, which may be substituted by 1-3 –OH groups, 1-3 halogen atoms, 1-3 haloalkyl groups (each having 1-3 halogen atoms), 1-3 O-alkyl groups, 1-3 –SH groups, 1-3 -S-alkyl groups, 1-3 -O-CO-alkyl groups, 1-3 -O-CO-NH-alkyl groups, 1-3 -NH-CO-alkyl groups, 1-3 -NH-CO-NH-alkyl groups, 1-3 -S(O) _n -alkyl groups, 1-3 -SO ₂ -NH-alkyl, 1-3 -NH-alkyl, 1-3 -N(alkyl) ₂ groups, 1-3 -NH((CH ₂ CH ₂ O) _1-20 H) groups, 1-3 -NH ₂ groups or 1-3 -(CH ₂ ) _0-3 Z groups, wherein Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ , wherein Y ¹ and Y ² independently of each other represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H, -(CH ₂ ) _0-3 -CH(NHCOCH ₃ )Z', -(CH ₂ ) _0-3 -CH(NH ₂ )Z' or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH (wherein "alkyl" is preferably C _1-10 -alkyl);

R ⁵ represents H, -MOD, NH ₂ , NO ₂ , halogen (especially F, Cl, Br), -CN, CF ₃ , -OCF ₃ , -CH ₂ F, -CH ₂ F, SH or -(CH ₂ ) _0-3 Z, wherein Z represents -H, -OY ³ , -SY ³ , halogen, NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH;

^R6 and ^R7 independently represent H or halogen (especially F, Cl, Br),

R ⁸ represents (optionally fluorinated) C _1-10 -alkyl;

wherein one or neither of the substituents R ¹ and R ³ represents –L-#1 or –L-BINDER,

L represents a linker and #1 represents a bond to a binder or a derivative thereof and BINDER represents a binder,

R ⁹ represents H, F, CH ₃ , CF ₃ , CH ₂ F or CHF ₂ ;

wherein —MOD represents —CH ₂ —S _x —(CH ₂ ) _0-4 -CHY ⁵ -COOH, wherein x is 0 or 1, and Y ⁵ represents H or NHY ⁶ , wherein Y ⁶ represents H or —COCH ₃ .

Also preferred are the following compounds, which may optionally be present with an acid, such as trifluoroacetic acid. These compounds may be linked to the binding entity via a linker at positions corresponding to those of R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁸ and R ¹⁰ , particularly R ¹ and R ³ (wherein a hydrogen atom is replaced by a linker):

N-(3-Aminopropyl)-N-{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide;

N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide;

(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-N-methylbutanamide (1:1);

N-(3-aminopropyl)-N-{(1S)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2yl]-2,2-dimethylpropyl}acetamide;

(15S,19R)-15-amino-19-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-18-glycoloyl-20,20-dimethyl-14-oxo-4,7,10-trioxa-13,18-diazaheneicosane-1-oic acid;

N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl-L-valyl-N ⁵ -carbamoyl-L-ornithine;

N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl-L-alanyl-N ⁵ -carbamoyl-L-ornithine;

N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl-4-amino-L-phenylalanine;

N-{(1R)-1-[1-(3-aminobenzyl)-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-N-(3-aminopropyl)-2-hydroxyacetamide (1:1);

(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butyric acid;

N-[(3S)-3-amino-4-hydrazino-4-oxobutyl]-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide (1:1);

N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-N-[(3S)-3,4-diaminobutyl]-2-hydroxyacetamide (1:1);

N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butyryl}-β-alanine;

(2S)-2-amino-N-(2-aminoethyl)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butanamide (2:1);

(1-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butanoyl}hydrazino)acetic acid;

N-[3-Amino-2-(sulfanylmethyl)propyl]-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide hydrochloride (1:1);

4-amino-N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}benzamide (2:1);

N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-N5-carbamoyl-L-ornithinamide (1:1);

L-valyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-N5-carbamoyl-L-ornithinamide (1:1);

L-valyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-N5-carbamoyl-L-ornithinamide (1:1);

N-(3-aminopropyl)-N-{(1S)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide;

S-(1-{2-[(N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl)amino]ethyl}-2,5-dioxopyrrolidin-3-yl)-L-cysteine;

S-[1-(2-{[2-({(2S)-2-amino-4-[{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]amino}-2-oxoethyl)-2,5-dioxopyrrolidin-3-yl]-L-cysteine;

N-{(2S)-2-amino-4-[{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl-L-alanyl-N-[4-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)phenyl]-N ⁵ -carbamoyl-L-ornithinamide;

S-(1-{2-[(N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl)amino]ethyl}-2,5-dioxopyrrolidin-3-yl)-L-cysteine;

S-[1-(2-{[2-({(2S)-2-amino-4-[{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]amino}-2-oxoethyl)-2,5-dioxopyrrolidin-3-yl]-L-cysteine;

N-{(2S)-2-amino-4-[{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl-L-alanyl-N-[4-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)phenyl]-N ⁵ -carbamoyl-L-ornithinamide;

S-(1-{2-[(N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl)amino]ethyl}-2,5-dioxopyrrolidin-3-yl)-L-cysteine;

N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexanoyl]-L-valyl-N ⁵ -carbamoyl-L-ornithine-N ⁶ -{(2S)-2-amino-4-[{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butyryl}-L-lysine;

S-[1-(2-{[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]amino}-2-oxoethyl)-2,5-dioxopyrrolidin-3-yl]-L-cysteine;

S-(2-{[2-({(2S)-2-amino-4-[{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]amino}-2-oxoethyl)-L-cysteine;

S-{1-[6-(2-{(2S)-2-amino-4-[{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}hydrazino)-6-oxohexyl]-2,5-dioxopyrrolidin-3-yl}-L-cysteine;

N-[19-(3(R/S)-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecan-1-yl]-R/S-{2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}homocysteine;

S-{(3R/S)-1-[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]-2,5-dioxopyrrolidin-3-yl}-L-cysteine;

N-[19-(3(R/S)-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecan-1-yl]-R/S-{2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}homocysteine;

S-[(3R/S)-1-(2-{[6-({2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}sulfanyl)hexanoyl]amino}ethyl)-2,5-dioxopyrrolidin-3-yl]-L-cysteine;

S-{1-[2-({[(1R,3S)-3-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)cyclopentyl]carbonyl}amino)ethyl]-2,5-dioxopyrrolidin-3-yl}-L-cysteine;

S-(2-{[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]amino}-2-oxoethyl)-L-cysteine;

N ⁶ -(N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-D-alanyl)-L-lysine;

N ⁶ -(N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl)-N ² -{N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexanoyl]-L-valyl-L-alanyl}-L-lysine;

N-[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]-L-glutamine;

N ⁶ -(N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butyryl}-β-alanyl)-L-lysine;

N-(3-Aminopropyl)-N-{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}acetamide;

N-(3-aminopropyl)-N-{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}-2-methoxyacetamide;

N-(3-aminopropyl)-N-{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}-2,4-difluorobenzamide;

N-(3-aminopropyl)-N-{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}-4-methylbenzamide;

N-(3-aminopropyl)-N-{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}-2-ethoxyacetamide;

N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-3,3,3-trifluoropropionamide;

N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-4-fluorobenzamide;

N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}acetamide;

N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-4-(trifluoromethyl)benzamide;

N-(3-aminopropyl)-N-{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}-2-ethoxyacetamide;

N-(3-aminopropyl)-N-{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}-2-ethoxyacetamide;

(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butanoic acid;

(2S)-2-amino-N-(2-aminoethyl)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butanamide;

4-[(2-{[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butanoyl}amino)ethyl]amino}-2-oxoethyl)amino]-3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-4-oxobutanoic acid;

4-[(2-{[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butanoyl}amino)ethyl]amino}-2-oxoethyl)amino]-2-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-4-oxobutanoic acid;

N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butyryl}-β-alanine;

N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-L-serine;

N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-L-alanine;

N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}glycine;

N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-4-methylbenzamide;

N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-4-(methylsulfanyl)benzamide;

(2S)-N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2-hydroxypropanamide;

N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2-(methylsulfanyl)acetamide;

(2S)-N-(3-aminopropyl)-N-{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}-2-hydroxypropanamide;

Methyl 4-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-4-oxobutanoate;

4-[(3-Aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-4-oxobutanoic acid;

(2R)-22-[(3R/S)-3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl]-2-[({2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}sulfanyl)methyl]-4,20-dioxo-7,10,13,16-tetraoxa-3,19-diazadocosan-1-oic acid;

4-amino-N-(3-aminopropyl)-N-{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}benzamide;

N-acetyl-S-{2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}-L-cysteine;

N-acetyl-S-[2-([3-(L-alanylamino)propyl]{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)-2-oxoethyl]-L-cysteine;

(2S)-N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}tetrahydrofuran-2-carboxamide;

3-({2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}sulfanyl)propanoic acid;

S-{2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}homocysteine;

4-amino-N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}benzamide;

4-[(2-{[(2R)-2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butanoyl}amino)-2-carboxyethyl]amino}-2-oxoethyl)amino]-3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-4-oxobutanoic acid;

4-[(2-{[(2R)-2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butanoyl}amino)-2-carboxyethyl]amino}-2-oxoethyl)amino]-2-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-4-oxobutanoic acid.

Particularly preferred according to the invention are compounds of the following formulae V, VI or VII, in which R ¹ , R ² , R ³ , R ⁴ and R ⁵ have the meanings mentioned above (as mentioned, for example, for formula (IIa) or (IIIa)):

Particularly preferred are compounds of formula V, VI, and VII, wherein ^R1 and ^R5 represent H or –L-#1; ^R2 and ^R4 independently represent –L-#1 or H; or ^R2 and ^R4 together (forming a pyrrolidine ring) represent _–CH2- ^CHR10- or ^–CHR10 - _CH2- , with ^R10 representing H or –L-#1; and ^R3 represents _CH2OH , CH( _CH3 )OH, or –L-#1, wherein one of the substituents ^R1 , ^R2 , ^R3 , ^R4 , ^R5 , and ^R10 represents –L-#1. Especially preferred are the corresponding compounds of formula VI.

Connector

The literature discloses various possibilities for covalently coupling (binding) organic molecules to binding entities, such as antibodies (see, for example, K. Lang and JW Chin. Chem. Rev. 2014, 114 , 4764-4806, M. Rashidian et al. Bioconjugate Chem. 2013, 24 , 1277-1294). According to the present invention, it is preferred that the KSP inhibitor be coupled to the antibody via one or more sulfur atoms of cysteine residues of the antibody, either already present as free thiols or generated by reduction of disulfide bridges, and/or via one or more NH groups of lysine residues of the antibody. However, the KSP inhibitor can also be linked to the antibody via a tyrosine residue, a glutamine residue, a residue of an unnatural amino acid, a free carboxyl group, or a carbohydrate residue of the antibody. For coupling, so-called linkers are used. Linkers can be classified into in vivo cleavable linkers and in vivo stable linkers (see L. Ducry and B. Stump, Bioconjugate Chem. 21 , 5-13 (2010)). In vivo cleavable linkers have a group that is cleavable in vivo, where a distinction can be made between groups that are chemically cleavable in vivo and groups that are enzymatically cleavable in vivo. "Chemically cleavable in vivo" and "enzymatically cleavable in vivo" mean that the linker or group is stable in the blood circulation and is only cleaved at or in the target cell by different chemical or enzymatic conditions (low pH; elevated glutathione concentration; the presence of lysosomal enzymes such as cathepsins or plasmin, or glycosidases (glycosidases), such as β-glucuronidase), thereby releasing the low molecular weight KSP inhibitor or its derivative. Groups that are chemically cleavable in vivo are particularly disulfides, hydrazones, acetals, and aminals; groups that are enzymatically cleavable in vivo are particularly 2-8-oligopeptide groups, especially dipeptide groups or glycosides. Peptide cleavage sites are disclosed in Bioconjugate Chem. 2002, 13, 855-869 and Bioorganic & Medicinal Chemistry Letters 8 (1998) 3341-3346 and Bioconjugate Chem. 1998, 9, 618-626. These include, for example, valine-alanine, valine-lysine, valine-citrulline, alanine-lysine and phenylalanine-lysine (optionally with an additional amide group).

In vivo stable linkers are characterized by high stability (less than 5% metabolites after 24 hours in plasma) and the absence of the aforementioned chemically or enzymatically cleavable in vivo groups.

The linker -L- preferably has one of the following basic structures (i) to (iv):

(i) –(CO) _m –SG1-L1-L2-

(ii) –(CO) _m –L1-SG-L1-L2-

(iii) –(CO) _m –L1-L2-

(iv) –(CO) _m –L1-SG-L2

Where m is 0 or 1; SG is a group that is cleavable in vivo (chemically or enzymatically) ( particularly a disulfide, hydrazone, acetal, or aminal; or a 2-8-mer oligopeptide group cleavable by cathepsins or plasmin); SG1 is an oligopeptide group or preferably a dipeptide group; L1 independently represents an in vivo stable organic group; and L2 represents a coupling group or a single bond to the conjugate. Conjugation is preferably to a cysteine or lysine residue of the conjugate. Alternatively, conjugation can be to a tyrosine residue, a glutamine residue, or an unnatural amino acid. Unnatural amino acids may contain, for example, an aldehyde or ketone group (e.g., formylglycine) or an azide or alkyne group (see Lan & Chin, Cellular Incorporation of Unnatural AminoAcids and Bioorthogonal Labeling of Proteins, Chem. Rev. 2014, 114, 4764-4806).

According to the present invention, the linker basic structure (iii) is particularly preferred, especially when the binding partner is an anti-TWEAKR antibody or an anti-EGFR antibody. Administration of the conjugate of the present invention having the linker basic structure (iii) and the linker coupled to a cysteine or lysine residue of the binding partner protein or peptide produces a cysteine or lysine derivative of the following formula through metabolism:

wherein L1 is in each case linked to a low molecular weight KSP inhibitor, for example a compound of formula (I), (IIa), (II), (III), (IIIa), (IIIb), (IIIc), (IIId), (IIIe) or (IV).

Also preferred according to the invention are the linker basic structures (ii) and (iv), in particular when attached at position (iii), in particular when the group L1 has one of the following structures:

(a) –NH-(CH ₂ ) _0-4- (CHCH ₃ ) _0-4 -CHY ⁵ -CO-Y ⁷ , wherein Y ⁵ represents H or NHY ⁶ , wherein Y ⁶ represents H or –COCH ₃ , and Y ⁷ represents a single bond or –NH-(CH ₂ ) _0-4 –CHNH ₂ -CO-, to obtain after cleavage the corresponding structure –NH-(CH ₂ ) _0-4- (CHCH ₃ ) _0-4 -CHY ⁵ -COOH or –NH-(CH ₂ ) _0-4- (CHCH ₃ ) _0-4 -CHY ⁵ -CO-NH-(CH ₂ ) _0-4 -CHNH ₂ -COOH.

(b) —CH ₂ —S _x —(CH ₂ ) _0-4 —CHY ⁵ —CO—, wherein x is 0 or 1, and Y ⁵ represents H or NHY ⁶ , wherein Y 6 represents H or —COCH ₃ , to obtain the corresponding structure —CH ₂ —S _x —(CH ₂ ) _0-4 —CHY ⁵ —COOH after cleavage.

This embodiment is preferred when L1 is in each case linked to a low molecular weight KSP inhibitor, for example a compound of formula (I), (IIa), (II), (III), (IIIa), (IIIb), (IIIc), (IIId), (IIIe) or (IV), in particular at position R ₄ . The binding entity is preferably an anti-TWEAKR antibody or an anti-EGFR antibody.

If the linker is attached to a cysteine side chain or cysteine residue, L2 is preferably derived from a group that reacts with the sulfhydryl group of cysteine. These include haloacetyl, maleimide, aziridine, acryloyl, arylated compounds, vinyl sulfone, pyridyl disulfide, TNB thiol, and disulfide-reducing agents. These groups typically react electrophilically with sulfhydryl bonds to form sulfide bridges (e.g., thioethers) or disulfide bridges. Stable sulfide bridges are preferred. L2 is preferably

in

# ¹ refers to the connection point with the sulfur atom of the bond.

# ² refers to the point of connection with the group L ¹ , and

R ₂₂ represents COOH, COOR, COR, CONHR, CONR ₂ (wherein R in each case represents C _1-3 -alkyl), CONH ₂ , preferably COOH.

L2 is particularly preferably:

or

wherein # ¹ refers to the point of attachment to the sulfur atom of the binding body, # ² refers to the point of attachment to the active substance, x represents 1 or 2, and R ²² represents COOH, COOR, COR, CONHR (wherein R represents C _1-3 -alkyl in each case), CONH ₂ , preferably COOH. Preferably, x=1 and R ²² represents COOH.

In the conjugates according to the invention or in the mixtures of the conjugates according to the invention, the bonds to the cysteine residues of the binder are present to an extent of preferably more than 80%, particularly preferably more than 90% (in each case based on the total number of bonds between the linker and the binder), particularly preferably as one of the two structures of the formula A3 or A4. In this case, the structures of the formula A3 or A4 are usually present together, preferably in a ratio of 60:40 to 40:60, based on the number of bonds to the binder. The remaining bonds are then present as the following structures:

.

According to the present invention, L1 is preferably represented by the following formula

in

R ¹⁰ represents H, NH ₂ or C ₁ -C ₃ -alkyl;

G1 represents -NHCO-, -CONH- or -NHCO-; (if G1 represents NHCO or -CONH-, then R ¹⁰ is preferably not NH ₂ ),

n is 0 or 1;

o is 0 or 1; and

G2 represents a linear or branched hydrocarbon chain having 1 to 100 carbon atoms, which is derived from arylene and/or linear and/or branched and/or cyclic alkylene and may be substituted by groups -O-, -S-, -SO-, SO ₂ , -NR ^y -, -NR ^y CO-, -C(NH)NR ^y -, CONR ^y -, -NR ^y NR ^y -, -SO ₂ NR ^y NR ^y -, -CONR ^y NR ^y - (wherein R ^y represents H, phenyl, C ₁ -C ₁₀ -alkyl, C ₂ -C ₁₀ -alkenyl or C ₂ -C ₁₀ -alkynyl, each of which may be substituted by NHCONH ₂ , -COOH, -OH, -NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid), -CO-, -CR ^x =NO- (wherein R ^x represents H, C ₁ -C ₃ -alkyl or phenyl) and/or one or more interrupted one or more times by a 3- to 10-membered aromatic or non-aromatic heterocyclic ring (preferably) having up to 4 heteroatoms selected from N, O and S, -SO- or -SO _{2 -} , wherein the hydrocarbon chain, including the side chains, if present, may be substituted by -NHCONH ₂ , -COOH, -OH, -NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid.

G2 represents a linear or branched hydrocarbon chain having 1 to 100 carbon atoms, which is derived from arylene and/or linear and/or branched and/or cyclic alkylene and may be interrupted one or more times by one or more of the radicals -O-, -S-, -SO-, SO ₂ , -NH-, -CO-, -NHCO-, -CONH-, -NMe-, -NHNH-, -SO ₂ NHNH-, -CONHNH- and a 5- to 10-membered aromatic or nonaromatic heterocycle (preferably) having up to 4 heteroatoms selected from N, O and S or -SO-, where side chains, if present, may be substituted by -NHCONH ₂ , -COOH, -OH, -NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid.

G2 preferably represents a linear or branched hydrocarbon chain having 1 to 100 carbon atoms, which is derived from arylene and/or linear and/or branched and/or cyclic alkylene and may be interrupted one or more times by one or more of the groups -O-, -S-, -SO-, SO2, _-NH- , -CO-, -NHCO-, -CONH-, -NMe-, _-NHNH- , -SO2NHNH-, -CONHNH-, ^-CRx =NO- (wherein ^Rx represents H, _C1 - _C3 -alkyl or phenyl) and a 3- to 10-membered, for example 5- to 10-membered, aromatic or non-aromatic heterocycle (preferably) having up to 4 heteroatoms selected from N, O and S, -SO- or _-SO2- , where the hydrocarbon chain, including side chains, if present, may be substituted by -NHCONH2, -COOH, _-OH , _-NH2 , NH- _CNNH2 , sulfonamide, sulfone, sulfoxide or sulfonic acid.

The further interrupting group in G2 is preferably

wherein Rx represents H, C ₁ -C ₃ -alkyl or phenyl.

Here, # ¹ is the bond to the KSP inhibitor and # ² is the bond to the coupling group (e.g., L2) of the binder.

The linear or branched hydrocarbon chain derived from the arylene group and/or the linear and/or branched and/or cyclic alkylene group generally contains an α,ω-divalent alkyl group with the number of carbon atoms indicated in each case. Preferred examples include: methylene, ethane-1,2-diyl (1,2-ethylene), propane-1,3-diyl (1,3-propylene), butane-1,4-diyl (1,4-butylene), pentane-1,5-diyl (1,5-pentylene), hexane-1,6-diyl (1,6-hexylene), heptane-1,7-diyl (1,7-hexylene), octane-1,8-diyl (1,8-octylene), nonane-1,9-diyl (1,9-nonylene), and decane-1,10-diyl (1,10-decylene). However, the alkylene groups in the hydrocarbon chain may also be branched, meaning that one or more hydrogen atoms of the aforementioned linear alkylene groups may optionally be replaced by C _1-10 alkyl groups, thereby forming side chains. The hydrocarbon chain may also contain cyclic alkylene groups (cycloalkanediyl groups), such as 1,4-cyclohexanediyl or 1,3-cyclopentanediyl. These cyclic groups may be unsaturated. In particular, aryl groups (arylene groups), such as phenylene groups, may be present in the hydrocarbon group. In the cyclic alkylene and arylene groups, one or more hydrogen atoms may also optionally be replaced by C _1-10 alkyl groups. This results in an optionally branched hydrocarbon chain. Such hydrocarbon chains have a total of 0 to 100 carbon atoms, preferably 1 to 50, and particularly preferably 2 to 25 carbon atoms.

If present, the side chain may be substituted with _-NHCONH2 , -COOH, -OH, _-NH2 , NH- _CNNH2 , sulfonamide, sulfone, sulfoxide, or sulfonic acid.

The hydrocarbon chain may be interrupted one or more times by one or more of the groups -O-, -S-, -SO-, SO ₂ , -NH-, -CO-, -NHCO-, -CONH-, -NMe-, -NHNH-, -SO ₂ NHNH-, -CONHNH- and a 5- to 10-membered aromatic or nonaromatic heterocyclic ring (preferred) having up to 4 heteroatoms selected from N, O and S, -SO- or -SO ₂ -.

The further interrupting group in G2 is preferably

.

The linker preferably conforms to the following formula:

§-(CO)m-L1-L2-§§

in

m is 0 or 1;

§ represents the bond to the active substance molecule and

§§ represents a bond to the binding partner peptide or protein, and

L1 and L2 have the meanings given above.

L1 particularly preferably has the formula -NR ¹¹ B-, wherein

R ¹¹ represents H or NH ₂ ;

B represents –[(CH ₂ ) _x -(X ⁴ ) _y ]w-(CH ₂ ) _z -,

w = 0 to 20;

x = 0 to 5;

x = 0 to 5;

y = 0 or 1;

z = 0 to 5; and

X ⁴ represents –O-, -CONH-, –NHCO- or .

Preferred linkers L according to the present invention have the following formula:

in

#3 represents the bond to the active substance molecule,

#4 represents the bond to the binding peptide or protein,

R ¹¹ represents H or NH ₂ ;

B represents –[(CH ₂ ) _x -(X ⁴ ) _y ] _w -(CH ₂ ) _z -,

w = 0 to 20;

x = 0 to 5;

y = 0 or 1;

z = 1 to 5; and

X ⁴ represents –O-, -CONH-, –NHCO- or .

The above-mentioned linker is particularly preferred in the conjugate of formula (I) or (II), wherein the linker is coupled by replacing the hydrogen atom at R ¹ or together with the cleavable linker SG1 at R ⁴ , that is, R ¹ represents -L-#1 or R ⁴ represents -SG1-L-#1, wherein #1 represents a bond to the conjugate.

Also preferred according to the invention are linkers in which, in the conjugate according to the invention or in the mixture of conjugates according to the invention, a bond to the cysteine residue of the binder is present to an extent of preferably more than 80%, particularly preferably more than 90% (in each case based on the total number of bonds of the linker to the binder), particularly preferably as one of the two structures of the formula A5 or A6:

in

# ¹ refers to the connection point with the sulfur atom of the bond.

# ² refers to the point of connection with the group L ¹ , and

R ²² represents COOH, COOR, COR, CONHR (wherein R in each case represents C 1-3 -alkyl), CONH ₂ , preferably COOH.

Here, the structures of the formula A5 or A6 are usually present together, preferably in a ratio of 60:40 to 40:60, based on the number of bonds to the combined body. The remaining bonds are then present as the following structure

.

Other linkers -L- attached to the cysteine side chain or cysteine residue have the following formula:

in

§ represents the bond to the active substance molecule and

§§ represents the bond to the binding peptide or protein,

m is 0, 1, 2, or 3;

n is 0, 1, or 2;

p is 0 to 20; and

L3 Representative

;

in

o is 0 or 1;

and

G ₃ represents a linear or branched hydrocarbon chain having 1 to 100 carbon atoms, which is derived from arylene and/or linear and/or cyclic alkylene and may be interrupted one or more times by one or more of the groups -O-, -S-, -SO-, SO ₂ , -NH-, -CO-, -NHCO-, -CONH-, -NMe-, -NHNH-, -SO ₂ NHNH-, -CONHNH- and a 3- to 10-membered (preferably 5- to 10-membered) aromatic or non-aromatic heterocycle (preferably) having up to 4 heteroatoms selected from N, O and S, -SO- or SO ₂ , where side chains, if present, may be substituted by -NHCONH ₂ , -COOH, -OH, -NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid.

In the above formula, preferably,

m is 1;

p is 0;

n is 0;

And L3 represents

;

in

o is 0 or 1; and

G ₃ represents -(CH ₂ CH ₂ O) _s (CH ₂ ) _t (CONH) _u CH ₂ CH ₂ O) _v (CH ₂ ) _w -, wherein

s, t, v and w are each independently 0 to 20, and u is 0 or 1.

Preferred radicals L1 in the above formula §-(CO)m-L1-L2-§§ are those shown below, where r in each case independently of one another represents a value of 0 to 20, preferably 0 to 15, particularly preferably 1 to 20, especially preferably 2 to 10:

Further examples of L1 are given in Table C, where this group is highlighted in a box.

Examples of linker moieties L1 are given in Tables A and A' below. The table also indicates which groups L2 these examples of L1 are preferably combined with, as well as the preferred coupling point ( ^R1 - ^R5 ) and the preferred value of m, i.e., whether a carbonyl group is present before L1 (see §-(CO)m-L1-L2-§§). These linkers are preferably coupled to cysteine residues. The first column also indicates the example number of a cetuximab ADC using the relevant linker, but this also applies to ADCs containing other antibodies in each row. If L2 is a succinimide or is derived therefrom, this imide can also be fully or partially in the form of the hydrolyzed open-chain succinamide as described above. Depending on L1, this hydrolysis to open-chain succinamide can be more or less pronounced, or not at all.

* ^R2 and ^R4 form a pyrrolidine ring substituted by a linker.

**Particularly preferably, the linker L1 given in these rows is linked to a linker L2 selected from the group consisting of:

and/or

Here, # ¹ refers to the point of attachment to the sulfur atom of the binder, # ² refers to the point of attachment to the group ^L1 , and ^R22 preferably represents COOH. In the conjugates or mixtures of conjugates according to the invention, bonds to the cysteine residue of the binder are present to an extent of preferably greater than 80%, particularly preferably greater than 90% (in each case based on the total number of bonds between the linker and the binder), particularly preferably as one of the two structures of formula A7 or A8. The structures of formula A7 or A8 are generally present together, preferably in a ratio of 60:40 to 40:60 based on the number of bonds to the binder. The remaining bonds are then present as the following structures:

.

**: See Note to Table A**

***: When this structure L2 is present, the following structure L2 may also be present:

.

Examples of conjugates with corresponding linkers have the following structures, wherein X1, X2, X3, and L1 have the meanings given above, L2 and L3 have the same meanings as L1, AK1 represents an antibody linked via a cysteine residue, and n is a number from 1 to 10. Particularly preferably, AK1 is a human, humanized, or chimeric monoclonal antibody or antigen-binding fragment thereof, in particular an anti-TWEAKR antibody or antigen-binding fragment thereof or an anti-EGFR antibody or antigen-binding fragment thereof. Particularly preferred are anti-TWEAKR antibodies that specifically bind to amino acid D (D47) in position 47 of TWEAKR (SEQ ID NO: 169), in particular the anti-TWEAKR antibody TPP-2090 or the anti-EGFR antibodies cetuximab or nimotuzumab.

If the linker is attached to a lysine side chain or a lysine residue, it preferably has the following formula:

-§-(SG) _x -L4-CO-§§

in

§ represents the bond to the active substance molecule and

§§ represents the bond to the binding peptide or protein,

x represents 0 or 1,

SG represents a cleavable group, preferably a 2-8 oligopeptide, particularly preferably a dipeptide,

and

L4 represents a single bond or a group -(CO) _y -G4-, wherein y represents 0 or 1, and

G4 represents a linear or branched hydrocarbon chain having 1 to 100 carbon atoms, which is derived from arylene and/or linear and/or branched and/or cyclic alkylene and may be interrupted one or more times by one or more of the radicals -O-, -S-, -SO-, SO ₂ , -NH-, -CO-, -NHCO-, -CONH-, -NMe-, -NHNH-, -SO ₂ NHNH-, -CONHNH- and a 5- to 10-membered aromatic or nonaromatic heterocycle (preferably) having up to 4 heteroatoms selected from N, O and S, -SO- or -SO 2 -, where side chains, if present, may be substituted by -NHCONH ₂ , -COOH, -OH, -NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid.

Table B below gives examples of linkers to lysine residues. The table also gives the preferred coupling points (R ¹ -R ⁵ ). The first column also indicates the example number in which the corresponding linker was used.

Table B: Lysine Linkers

-§-(SG) _x -L4-CO-§§

.

Examples of conjugates with corresponding linkers have the following structures, wherein X1, X2, X3, and L4 have the meanings given above, AK2 represents an antibody linked via a lysine residue, and n is a number from 1 to 10. Particularly preferably, AK2 is a human, humanized, or chimeric monoclonal antibody or antigen-binding fragment thereof, in particular an anti-TWEAKR antibody or antigen-binding fragment thereof or an anti-EGFR antibody or antigen-binding fragment thereof. Particularly preferred are anti-TWEAKR antibodies that specifically bind to amino acid D (D47) in position 47 of TWEAKR (SEQ ID NO: 169), in particular the anti-TWEAKR antibody TPP-2090 or the anti-EGFR antibodies cetuximab or nimotuzumab.

Also preferred according to the invention are basic structures (i), (ii) or (iv) in which SG1 or SG represents a group cleavable by cathepsins and L1 and L2 have the meanings given above. Particularly preferred are the following groups:

-Val-Ala-CONH- (this cleaves the amide bond at the C-terminal amide of alanine)

-NH-Val-Lys-CONH- (cleaves the amide bond at the C-terminal amide of lysine)

-NH-Val-Cit-CONH- (cleaves the amide bond at the C-terminal amide of citrulline)

-NH-Phe-Lys-CONH (cleaves the amide bond at the C-terminal amide of lysine)

-NH-Ala-Lys-CONH- (cleaves the amide bond at the C-terminal amide of lysine)

-NH-Ala-Cit-CONH- (cleaves the amide bond at the C-terminal amide of citrulline)

SG1 or SG is particularly preferably

or

or

wherein X represents H or C _1-10 -alkyl which may be optionally substituted by —NHCONH ₂ , —COOH, —OH, NH ₂ , —NH-CNNH ₂ or sulfonic acid.

Table C below provides examples of linker moieties –SG1-L1- or –L1-SG-L1-, where SG1 and SG are groups cleavable by cathepsins. Table C also indicates which groups L2 these examples of –SG1-L1- and –L1-SG-L1- are preferably combined with, as well as the preferred coupling point (R ¹ -R ⁵ ) and the preferred value of m, i.e., whether a carbonyl group is present before L1 (see §-(CO)m-L1-L2-§§). These linkers are preferably coupled to cysteine residues. The first column also provides example numbers for cetuximab ADCs using the corresponding linkers, as shown. These are also applicable to corresponding ADCs containing other antibodies. The L1 group is highlighted in a box. However, these L1 groups can be replaced by one of the L1 groups given for the formula §-(CO)m-L1-L2-§§ above. If L2 is succinamide or derived therefrom, this amide can also be fully or partially in the form of the hydrolyzed open-chain succinamide as described above.

An example of a conjugate having the basic structure (i) has the following structure, wherein X1, X2 and X3 have the meanings given above, L4 has the same meaning as L1, AK1 represents an antibody linked via a cysteine residue, and n is a number from 1 to 10. Particularly preferably, AK1 is an anti-TWEAKR antibody, in particular an anti-TWEAKR antibody that specifically binds to amino acid D (D47) in position 47 of TWEAKR (SEQ ID NO: 169), in particular the anti-TWEAKR antibody TPP-2090.

Preparation of KSP inhibitor-linker intermediates and conjugates

The conjugates of the present invention are prepared by first providing a low molecular weight KSP inhibitor containing a linker. The intermediate thus obtained is then reacted with a binding partner, preferably an antibody.

Preferably, for coupling to cysteine residues, one of the following compounds is reacted with a cysteine-containing binding entity, such as an antibody, which is optionally partially reduced for this purpose:

Where R represents -H or –COOH,

wherein K represents a linear or branched C 1 -C ₆ alkyl group optionally substituted by a C _{1 -C 6} -alkoxy group or -OH, and

wherein X ₁ , X ₂ , X ₃ , SG1 , L1 , L2 , L3 and L4 have the same meanings as described above.

The compound can be used, for example, in the form of its trifluoroacetate salt. For reactions with binding partners, such as antibodies, the compound is preferably used in a 2- to 12-fold molar excess relative to the binding partner.

Preferably, for coupling to lysine residues, one of the following compounds is reacted with a lysine-containing binding agent, such as an antibody:

wherein X ₁ , X ₂ , and X ₃ have the same meanings as in formula (II), and L4 has the same meaning as L1, and L1 has the same meaning as described above.

For coupling to an intermediate on a cysteine residue, the reaction can be performed as follows:

Other intermediates and other antibodies can be reacted accordingly.

For coupling to the intermediate on the lysine group, the reaction can be as follows:

According to the invention, this results in the following conjugate:

Depending on the linker, succinimide-linked ADCs can be converted after conjugation to open-chain succinamides with a favorable stability profile.

This reaction (ring opening) can be carried out, for example, by stirring, at a pH of 7.5 to 9, preferably at a pH of 8, at a temperature of 25° C. to 37° C. The stirring time is preferably 8 to 30 hours.

In the above formula, _X1 , _X2 , and _X3 have the same meanings as in formula (II), SG1 and L1 have the same meanings as described above, and L2, L3, and L4 have the same meanings as L1; R and K have the same meanings as described above. AK1 is an antibody conjugated via a cysteine residue, and AK2 is an antibody conjugated via a lysine residue. Particularly preferably, AK1 and AK2 are anti-TWEAKR antibodies, particularly antibodies that specifically bind to amino acid D (D47) at position 47 of TWEAKR (SEQ ID NO: 169), particularly the anti-TWEAKR antibody TPP-2090.

combination

In the broadest sense, the term "binding agent" is understood to refer to a molecule that binds to a target molecule present on a certain target cell population to which the binding agent/active substance conjugate is directed. The term binding agent should be understood in its broadest sense and also includes, for example, lectins, proteins capable of binding to certain sugar chains, and phospholipid-binding proteins. Such binding agents include, for example, high molecular weight proteins (binding proteins), polypeptides or peptides (binding peptides), non-peptides (such as aptamers (US Pat. No. 5,270,163) (reviewed by Keefe AD. et al., Nat. Rev. Drug Discov. 2010;9:537-550) or vitamins), and all other cell-binding molecules or substances. Binding proteins are, for example, antibodies and antibody fragments or mimetics, such as affibodies, adnectins, anticalins, DARPins, avimers, nanobodies (reviewed by Gebauer M. et al., Curr. Opinion in Chem. Biol. 2009; 13: 245-255; Nuttall S.D. et al., Curr. Opinion in Pharmacology 2008; 8: 608-617). Binding peptides are, for example, ligands of a ligand/receptor pair, such as VEGF of the ligand/receptor pair VEGF/KDR, transferrin of the ligand/receptor pair transferrin/transferrin receptor, or cytokine/cytokine receptor, such as TNFα of the ligand/receptor pair TNFα/TNFα receptor.

The literature also discloses various possibilities for covalently coupling (binding) organic molecules to antibodies. According to the present invention, it is preferred that the toxin cluster be coupled to the antibody via one or more sulfur atoms of cysteine residues and/or via one or more NH groups of lysine residues. However, the toxin cluster can also be attached to the antibody via free carboxyl groups or via carbohydrate residues of the antibody.

"Target molecule" is understood in the broadest sense to mean a molecule present in the target cell population and can be a protein (e.g. a receptor for a growth factor) or a non-peptide molecule (e.g. a sugar or phospholipid). It is preferably a receptor or an antigen.

The term "extracellular" target molecule describes a target molecule located outside the cell that is attached to the cell, or a portion of a target molecule located outside the cell. That is, a binding partner can attach to its extracellular target molecule in an intact cell. Extracellular target molecules can be anchored in the cell membrane or be a component of the cell membrane. Those skilled in the art are aware of methods for identifying extracellular target molecules. For proteins, this can be accomplished by determining the transmembrane domain(s) and the orientation of the protein within the membrane. This data is typically stored in protein databases (e.g., SwissProt).

The term "cancer target molecule" describes a target molecule that is present in greater amounts on one or more cancer cell types compared to non-cancerous cells of the same tissue type. Preferably, the cancer target molecule is selectively present on one or more cancer cell types compared to non-cancerous cells of the same tissue type, wherein "selective" describes at least a two-fold enrichment on cancer cells compared to non-cancerous cells of the same tissue type ("selective cancer target molecule"). The use of a cancer target molecule allows for the selective treatment of cancer cells by the conjugates of the present invention.

The binding agent can be linked to the linker via a bond. The binding agent can be linked via heteroatoms of the binding agent. Heteroatoms of the binding agent that can be used for linking according to the present invention are sulfur (in one embodiment, via a sulfhydryl group of the binding agent), oxygen (in one embodiment, via a carboxyl or hydroxyl group of the binding agent), and nitrogen (in one embodiment, via a primary or secondary amine or amide group of the binding agent). These heteroatoms can be present in the natural binding agent or introduced by chemical methods or molecular biological methods. According to the present invention, the connection of the binding agent to the toxin cluster has only a slight effect on the binding activity of the binding agent to the target molecule. In a preferred embodiment, the connection has no effect on the binding activity of the binding agent to the target molecule.

According to the present invention, the term "antibody" should be understood in its broadest sense and includes immunoglobulin molecules, such as complete or modified monoclonal antibodies, polyclonal antibodies, or multispecific antibodies (e.g., bispecific antibodies). An immunoglobulin molecule preferably comprises four polypeptide chains, two heavy chains (H chains) and two light chains (L chains), typically connected by disulfide bridges. Each heavy chain comprises a heavy chain variable domain (abbreviated as VH) and a heavy chain constant domain. The heavy chain constant domain may, for example, comprise three domains: CH1, CH2, and CH3. Each light chain comprises a variable domain (abbreviated as VL) and a constant domain. The light chain constant domain comprises a structural domain (abbreviated as CL). The VH and VL domains can be further subdivided into hypervariable regions, also known as complementarity determining regions (CDRs), and regions of low sequence variability (framework regions, abbreviated as FRs). Typically, each VH and VL region consists of three CDRs and up to four FRs. For example, from amino-terminus to carboxyl-terminus, the following order is used: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The antibody can be obtained from any suitable species, such as rabbit, llama, camel, mouse or rat. In one embodiment, the antibody is of human or murine origin. The antibody can, for example, be human, humanized or chimeric.

The term "monoclonal" antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies in the population are identical except for naturally occurring mutations, which may be minor. A monoclonal antibody recognizes a single antigen-binding site with high specificity. The term monoclonal antibody does not refer to a particular method of production.

The term "complete" antibody refers to an antibody that comprises an antigen binding domain and the constant domains of both the light and heavy chains. The constant domains may be naturally occurring domains or variants having a number of modified amino acid positions.

The term "modified complete antibody" refers to a complete antibody that is fused to another polypeptide or protein not derived from the antibody via its amino or carboxyl terminus via a covalent bond (e.g., a peptide bond). In addition, antibodies can be modified to introduce reactive cysteines at specific positions to facilitate coupling to toxins (see Junutula et al. Nat Biotechnol. 2008 Aug; 26(8): 925-32).

The term "human" antibody refers to an antibody that is obtainable from a human or a synthetic human antibody. A "synthetic" human antibody is an antibody that is partially or, more difficultly, completely obtainable by computer simulation of a synthetic sequence based on analysis of human antibody sequences. A human antibody can, for example, be encoded by a nucleic acid isolated from a library of human antibody sequences. Examples of such antibodies can be found in Söderlind et al., Nature Biotech. 2000, 18:853-856.

The term "humanized" or "chimeric" antibody describes an antibody composed of a non-human portion of the sequence and a human portion. In these antibodies, a portion of the sequence of a human immunoglobulin (the acceptor) is replaced by a portion of the sequence of a non-human immunoglobulin (the donor). In many cases, the donor is a murine immunoglobulin. In the case of a humanized antibody, the amino acids in the CDRs of the acceptor are replaced by amino acids from the donor. Sometimes, the amino acids in the framework are also replaced by the corresponding amino acids from the donor. In some cases, the humanized antibody contains amino acids that are introduced during the optimization process of the antibody and are not contained in either the acceptor or the donor. In the case of a chimeric antibody, the variable domains of the donor immunoglobulin are fused to the constant regions of a human antibody.

The term complementary determining region (CDR) as used herein refers to the amino acids of the antibody variable region required for binding to the antigen. Typically, each variable region has three CDR regions, referred to as CDR1, CDR2, and CDR3. Each CDR region may include amino acids according to the Kabat definition and/or amino acids according to the hypervariable loops (loops) defined by Chotia. According to Kabat's definition, the region comprising, for example, amino acid positions 24-34 (CDR1), 50-56 (CDR2), and 89-97 (CDR3) from approximately the variable light chain and 31-35 (CDR1), 50-65 (CDR2), and 95-102 (CDR3) from the variable heavy chain (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The definition according to Chotia includes, for example, a region from approximately amino acid positions 26-32 (CDR1), 50-52 (CDR2), and 91-96 (CDR3) of the variable light chain and 26-32 (CDR1), 53-55 (CDR2), and 96-101 (CDR3) of the variable heavy chain (Chothia and Lesk; J Mol Biol 196: 901-917 (1987)). In some cases, a CDR may include amino acids from a CDR region according to the definitions of Kabat and Chotia.

Antibodies can be divided into different classes based on the amino acid sequence of their heavy chain constant domain. There are five major classes of complete antibodies: IgA, IgD, IgE, IgG, and IgM, several of which are further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to these classes are called [alpha/α], [delta/δ], [epsilon/ε], [gamma/γ], and [my/μ]. The three-dimensional structure and subunit structure of antibodies are both known.

The term "functional fragment" or "antigen-binding antibody fragment" of an antibody/immunoglobulin is defined as a fragment of an antibody/immunoglobulin that still contains the antigen-binding domain of the antibody/immunoglobulin (e.g., the variable domain of IgG). The "antigen-binding domain" of an antibody typically comprises one or more hypervariable regions of the antibody, such as the CDR, CDR2, and/or CDR3 regions. However, the "framework" or "skeleton" region of the antibody may also play a role in binding the antibody to the antigen. The framework region forms the backbone of the CDRs. The antigen-binding domain preferably comprises at least amino acids 4 to 103 of the variable light chain and amino acids 5 to 109 of the variable heavy chain, more preferably amino acids 3 to 107 of the variable light chain and amino acids 4 to 111 of the variable heavy chain, and particularly preferably the complete variable light and heavy chains, i.e., amino acids 1–109 of VL and 1 to 113 of VH (numbering according to WO 97/08320).

"Functional fragments" or "antigen-binding antibody fragments" of the present invention include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, diabodies, single-domain antibodies (DAb), linear antibodies, single-chain antibodies (single-chain Fv, abbreviated as scFv); and multispecific antibodies, such as bispecific and trispecific antibodies, formed, for example, from antibody fragments C. A. K Borrebaeck, ed. (1995) Antibody Engineering (Breakthroughs in Molecular Biology), Oxford University Press; R. Kontermann & S. Duebel, eds. (2001) Antibody Engineering (Springer Laboratory Manual), Springer Verlag. Antibodies that are not "multispecific" or "multifunctional" antibodies are those that have the same binding site. Multispecific antibodies can be specific for different epitopes of an antigen or can be specific for epitopes of more than one antigen (see, for example, WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt et al., 1991, J. Immunol. 147:60-69; U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; or Kostelny et al., 1992, J. Immunol. 148:1547-1553). F(ab')2 or Fab molecules can be constructed to reduce or completely prevent the number of intermolecular disulfide interactions that occur between the C1 and CL domains.

"Epitope" refers to a protein determinant that can specifically bind to an immunoglobulin or T-cell receptor. Epitope determinants are usually composed of chemically active surface groups of molecules such as amino acids or sugar side chains, or combinations thereof, and usually have specific three-dimensional structural properties as well as specific charge properties.

"Functional fragments" or "antigen-binding antibody fragments" can be fused to another polypeptide or protein not derived from the antibody via a covalent bond (e.g., a peptide bond) at their amino or carboxyl termini. In addition, antibodies and antigen-binding fragments can be modified by introducing reactive cysteines at specific positions to facilitate coupling to the toxin cluster (see Junutula et al. Nat Biotechnol. 2008 Aug; 26(8): 925-32).

Polyclonal antibodies can be prepared by methods known to those skilled in the art. Monoclonal antibodies can be prepared by methods known to those skilled in the art (Köhler and Milstein, Nature, 256, 495-497, 1975). Human and humanized monoclonal antibodies can be prepared by methods known to those skilled in the art (Olsson et al., Meth Enzymol. 92, 3-16 or Cabilly et al., US Pat. No. 4,816,567 or Boss et al., US Pat. No. 4,816,397).

Those skilled in the art are aware of various methods for preparing human antibodies and fragments thereof, for example, using transgenic mice (N Lonberg and D Huszar, Int Rev Immunol. 1995; 13(1): 65-93) or phage display technology (Clackson et al., Nature. 1991 Aug 15; 352(6336): 624-8). The antibodies of the present invention can be obtained, for example, from a recombinant antibody library composed of the amino acid sequences of a diverse range of antibodies collected from a large number of healthy volunteers. Antibodies can also be produced using known recombinant DNA techniques. The nucleic acid sequence of the antibody can be obtained by conventional sequencing or can be obtained from publicly accessible databases.

"Isolated" antibodies or binding bodies have been purified to remove other components of cells. Contaminating components that may interfere with the cell for diagnosis or therapeutic purposes are, for example, other peptides or non-peptide components of enzymes, hormones or cells. Preferred antibodies or binding bodies have been purified to a degree greater than 95 % by weight of the antibody or binding body (for example, by Lowry method, UV-Vis spectroscopy or by SDS capillary gel electrophoresis). In addition, the antibody has been purified to at least 15 amino acids that can determine the aminoterminal or internal amino acid sequence or has been purified to homogeneity, wherein homogeneity is measured by SDS-PAGE under reducing or non-reducing conditions (can be detected by Coomassie blue staining or preferably by silver staining). However, antibodies are usually prepared by one or more purification steps.

The term "specific binding" refers to the binding of an antibody or binding entity to a predetermined antigen/target molecule. Specific binding of an antibody or binding entity generally describes an antibody or binding entity with an affinity of at least 10 ^{^-7} M (as a Kd value; i.e., ^preferably those with Kd values less than 10^-7 M) , wherein the affinity of the antibody or binding entity for the predetermined antigen/target molecule is at least two-fold greater than for a nonspecific antigen/target molecule that is not the predetermined antigen/target molecule or a closely related antigen/target molecule (e.g., bovine serum albumin or casein). The antibody preferably has an affinity of at least 10 ^{^-7} M (as a Kd value; in other words, preferably those with Kd values less than 10 ^{^-7} M), preferably at least 10 ^{^-8} M, and more preferably between 10 ^{^-9} M and 10 ^{^-11} M. The Kd value can be determined, for example, by surface plasmon resonance spectroscopy.

The antibody/active substance conjugates of the present invention also exhibit affinities within these ranges. This affinity is preferably not substantially affected by the conjugation of the active substance (generally, the affinity is reduced by less than one order of magnitude, in other words, for example, from 10 ⁻⁸ M to 10 ⁻⁷ M at most).

Antibodies used according to the present invention are also preferably characterized by high selectivity. High selectivity is present when an antibody of the present invention exhibits an affinity for the target protein that is at least 2-fold, preferably 5-fold, or more preferably 10-fold better than for an independent antigen, such as human serum albumin (affinity can be determined, for example, by surface plasmon resonance spectroscopy).

Furthermore, the antibodies of the present invention used are preferably cross-reactive. To facilitate and better interpret preclinical studies, such as toxicology or efficacy studies (e.g., in xenograft mice), it is advantageous if the antibodies used according to the present invention bind not only to the human target protein but also to a species target protein from the species being studied. In one embodiment, the antibodies used according to the present invention are cross-reactive against target proteins from at least one other species in addition to the human target protein. For toxicology and/or efficacy studies, species from the families Rodentia, Canidae, and Non-human Primates are preferably used. Preferred rodent species are mice and rats. Preferred non-human primates are Macaques, Orangutans, and Long-tailed Macaques.

In one embodiment, the antibodies used according to the present invention are cross-reactive, in addition to the human target protein, to a target protein of at least one other species selected from mouse, rat, and long-tailed macaque (cynomolgus monkey). Particularly preferred are antibodies used according to the present invention that are cross-reactive, in addition to the human target protein, to at least a mouse target protein. Preferred are cross-reactive antibodies whose affinity for the target protein of another non-human species differs by no more than 50 times, and in particular no more than 10 times, from their affinity for the human target protein.

Antibodies against cancer targets

The target molecule to which a binding entity, such as an antibody or antigen-binding fragment thereof, is directed is preferably a cancer target molecule. The term "cancer target molecule" describes a target molecule that is present in greater amounts on one or more cancer cell types compared to non-cancerous cells of the same tissue type. Preferably, the cancer target molecule is selectively present on one or more cancer cell types compared to non-cancerous cells of the same tissue type, wherein "selective" describes at least a two-fold enrichment on cancer cells compared to non-cancerous cells of the same tissue type ("selective cancer target molecule"). The use of a cancer target molecule allows for the selective treatment of cancer cells by the conjugates of the present invention.

Antibodies specific for antigens, such as cancer cell antigens, can be prepared by one of ordinary skill in the art using methods familiar to him or her (e.g., recombinant expression) or are commercially available (e.g., from Merck KGaA, Germany). Examples of known commercially available antibodies for use in cancer therapy include Erbitux® (cetuximab, Merck KGaA), Avastin® (bevacizumab, Roche), and Herceptin® (trastuzumab, Genentech). Trastuzumab is a recombinant humanized monoclonal antibody of the IgG1κ type that binds to the extracellular domain of the human epidermal growth receptor with high affinity (Kd = 5 nM) in cell-based assays. This antibody is recombinantly produced in CHO cells.

In a preferred embodiment, the target molecule is a selective cancer target molecule.

In a particularly preferred embodiment, the target molecule is a protein.

In one embodiment, the target molecule is an extracellular target molecule.In a preferred embodiment, the extracellular target molecule is a protein.

Cancer target molecules are known to those skilled in the art. Examples of these are listed below.

Examples of cancer target molecules are:

(1) EGF receptor (NCBI reference sequence NP_005219.2), SEQ ID NO: 213 (1210 amino acids):

The extracellular domain is marked by underlining.

(2) Mesothelin (SwissProt reference Q13421-3), SEQ ID NO: 214 (622 amino acids):

>sp|Q13421-3|MSLN_HUMAN isoform 2 of mesothelin OS=Homo sapiens GN=MSLN

Mesothelin is encoded by amino acids 296-598, while amino acids 37-286 encode megakaryocyte-enhancing factor. Mesothelin is anchored to the cell membrane via a GPI anchor and localized extracellularly.

(3) Carbonic anhydrase IX (SwissProt reference Q16790), SEQ ID NO: 215 (459 amino acids):

>sp|Q16790|CAH9_HUMAN Carbonic Anhydrase 9 OS=Homo sapiens GN=CA9 PE=1 SV=2

The extracellular domain is marked by underlining.

(4) C4.4a (NCBI reference sequence NP_055215.2; synonym LYPD3), SEQ ID NO: 216 (346 amino acids):

>gi|93004088|ref|NP_055215.2|ly6/PLAUR domain-containing protein 3 - precursor [Homo sapiens]

The mature ectodomain is marked by underlining.

(5) CD52 (NCBI reference sequence NP_001794.2), SEQ ID NO: 217

>gi|68342030|ref|NP_001794.2| CAMPATH-1 antigen precursor [Homo sapiens]

(6) Her2 (NCBI reference sequence NP_004439.2), SEQ ID NO: 218

>gi|54792096|ref|NP_004439.2|Receptor tyrosine-protein kinase erbB-2 isoform a [Homo sapiens]

(7) CD20 (NCBI reference sequence NP_068769.2), SEQ ID NO: 219

>gi|23110987|ref|NP_068769.2| B-lymphocyte antigen CD20 [Homo sapiens]

(8) Lymphocyte activation antigen CD30 (SwissProt ID P28908), SEQ ID NO: 220

>gi|68348711|ref|NP_001234.2| Tumor necrosis factor receptor superfamily member 8 isoform 1-precursor [Homo sapiens]

(9) Lymphocyte adhesion molecule CD22 (SwissProt ID P20273), SEQ ID NO: 221

>gi|157168355|ref|NP_001762.2| B-cell receptor CD22 isoform 1-precursor [Homo sapiens]

(10) Myeloid cell surface antigen CD33 (SwissProt ID P20138), SEQ ID NO: 222

>gi|130979981|ref|NP_001763.3| Myeloid cell surface antigen CD33 isoform 1-precursor [Homo sapiens]

(11) Transmembrane glycoprotein NMB (SwissProt ID Q14956), SEQ ID NO: 223

>gi|52694752|ref|NP_001005340.1| Transmembrane glycoprotein NMB isoform alpha-precursor [Homo sapiens]

(12) Adhesion molecule CD56 (SwissProt ID P13591), SEQ ID NO: 224

>gi|94420689|ref|NP_000606.3|Neural cell adhesion molecule 1 isoform 1 [Homo sapiens]

(13) Surface molecule CD70 (SwissProt ID P32970), SEQ ID NO: 225

>gi|4507605|ref|NP_001243.1| CD70 antigen [Homo sapiens]

(14) Surface molecule CD74 (SwissProt ID P04233), SEQ ID NO: 226

>gi|10835071|ref|NP_004346.1| HLA class II histocompatibility antigen gamma chain isotype b [Homo sapiens]

(15) B-lymphocyte antigen CD19 (SwissProt ID P15391), SEQ ID NO: 227

>gi|296010921|ref|NP_001171569.1| B-lymphocyte antigen CD19 isoform 1-precursor [Homo sapiens]

(16) Surface protein mucin-1 (SwissProt ID P15941), SEQ ID NO: 228

>gi|65301117|ref|NP_002447.4| Mucin-1 isoform 1-precursor [Homo sapiens]

(17) Surface protein CD138 (SwissProt ID P18827), SEQ ID NO: 229

>gi|29568086|ref|NP_002988.3|Syndecan-1-precursor [Homo sapiens]

(18) Integrin αV (Genbank Accession No.: NP_002201.1), SEQ ID NO: 230

>gi|4504763|ref|NP_002201.1| Integrin alpha-V isoform 1 - precursor [Homo sapiens]

(19) Teratoma-derived growth factor 1 protein TDGF1 (Genbank Accession No.: NP_003203.1), SEQ ID NO: 231

>gi|4507425|ref|NP_003203.1| Teratoma-derived growth factor 1 isoform 1-precursor [Homo sapiens]

(20) Prostate-specific membrane antigen PSMA (Swiss Prot ID: Q04609), SEQ ID NO: 232

>gi|4758398|ref|NP_004467.1| Glutamate carboxypeptidase 2 isoform 1 [Homo sapiens]

(21) Tyrosine protein kinase EPHA2 (Swiss Prot ID: P29317), SEQ ID NO: 233

>gi|32967311|ref|NP_004422.2| Ephrin type A receptor 2-pro-form [Homo sapiens]

(22) Surface protein SLC44A4 (Genbank Accession No: NP_001171515), SEQ ID NO: 234

>gi|295849282|ref|NP_001171515.1|Choline transporter-like protein 4 isoform 2 [Homo sapiens]

(23) Surface protein BMPR1B (SwissProt: O00238)

(24) Transport protein SLC7A5 (SwissProt: Q01650)

(25) Epithelial prostate antigen STEAP1 (SwissProt: Q9UHE8)

(26) Ovarian cancer antigen MUC16 (SwissProt: Q8WXI7)

(27) Transport protein SLC34A2 (SwissProt: O95436)

(28) Surface protein SEMA5b (SwissProt: Q9P283)

(29) Surface protein LYPD1 (SwissProt: Q8N2G4)

(30) Endothelin receptor type B EDNRB (SwissProt: P24530)

(31) RING finger protein RNF43 (SwissProt: Q68DV7)

(32) Prostate cancer-related protein STEAP2 (SwissProt: Q8NFT2)

(33) Cation channel TRPM4 (SwissProt: Q8TD43)

(34) Complement receptor CD21 (SwissProt: P20023)

(35) B cell antigen receptor complex-associated protein CD79b (SwissProt: P40259)

(36) Cell adhesion antigen CEACAM6 (SwissProt: P40199)

(37) Dipeptidase DPEP1 (SwissProt: P16444)

(38) Interleukin receptor IL20Rα (SwissProt: Q9UHF4)

(39) Proteoglycan BCAN (SwissProt: Q96GW7)

(40) Ephrin receptor EPHB2 (SwissProt: P29323)

(41) Prostate stem cell-associated protein PSCA (Genbank Accession No: NP_005663.2 )

(42) Surface protein LHFPL3 (SwissProt: Q86UP9)

(43) Receptor protein TNFRSF13C (SwissProt: Q96RJ3)

(44) B cell antigen receptor complex-associated protein CD79a (SwissProt: P11912)

(45) Receptor protein CXCR5 (SwissProt: P32302)

(46) Ion channel P2X5 (SwissProt: Q93086)

(47) Lymphocyte antigen CD180 (SwissProt: Q99467)

(48) Receptor protein FCRL1 (SwissProt: Q96LA6)

(49) Receptor protein FCRL5 (SwissProt: Q96RD9)

(50) MHC class II molecule Ia antigen HLA-DOB (Genbank Accession No: NP_002111.1)

(51) T cell protein VTCN1 (SwissProt: Q7Z7D3)

(52) TWEAKR (SEQ ID NO: 169 (protein); SEQ ID NO: 170 (DNA).

(53) Lymphocyte antigen CD37 (Swiss Prot: P11049)

(54) FGF receptor 2; FGFR2 (Gene ID: 2263; Gene name: FGFR2). This FGFR2 receptor exists in different splice variants (α, β, IIIb, IIIc). All splice variants can serve as target molecules;

(55) Transmembrane glycoprotein B7H3 (CD276; Gene ID: 80381)

(56) B cell receptor BAFFR (CD268; Gene ID: 115650)

(57) Receptor protein ROR 1 (Gene ID: 4919)

(58) Surface receptor IL3RA (CD123; Gene ID: 3561)

(59) CXC chemokine receptor CXCR5 (CD185; gene ID 643)

(60) Receptor protein syncytin (Gene ID 30816).

In a preferred subject matter of the present invention, the cancer target molecule is selected from the group consisting of cancer target molecules (1) to (60), in particular (1), (6) and (52).

In another particularly preferred subject matter of the present invention, the binding entity binds to an extracellular cancer target molecule selected from the group consisting of cancer target molecules (1) to (60), in particular (1), (6) and (52).

In another particularly preferred subject matter of the present invention, the binding body specifically binds to an extracellular cancer target molecule selected from the group consisting of cancer target molecules (1) to (60), in particular (1), (6) and (52). In a preferred embodiment, the binding body is internalized by the target cell after binding to the extracellular target molecule on the target cell. This results in the binding body/active substance conjugate (which may be an immunoconjugate or ADC) being taken up by the target cell. The binding body is then preferably processed intracellularly, preferably by means of lysosomes.

In one embodiment, the binding entity is a binding protein. In a preferred embodiment, the binding entity is an antibody, an antigen-binding antibody fragment, a multispecific antibody, or a mimetic antibody.

Preferred mimetics are Affibodies, Adnectins, Anticalins, DARPins, Avimers or Nanobodies. Preferred multispecific antibodies are bispecific and trispecific antibodies.

In a preferred embodiment, the binding entity is an antibody or an antigen-binding antibody fragment, more preferably an isolated antibody or an isolated antigen-binding antibody fragment.

Preferred antigen-binding antibody fragments are Fab, Fab', F(ab')2 and Fv fragments, diabodies, DAbs, linear antibodies and scFv. Fab, diabodies and scFv are particularly preferred.

In a particularly preferred embodiment, the binding entity is an antibody. Particularly preferred is a monoclonal antibody or an antigen-binding antibody fragment thereof. Even more particularly preferred is a human, humanized, or chimeric antibody or an antigen-binding antibody fragment thereof.

Antibodies or antigen-binding antibody fragments that bind to cancer target molecules can be prepared by one of ordinary skill in the art using known methods, such as chemical synthesis or recombinant expression. Binding agents to cancer target molecules are commercially available or can be prepared by one of ordinary skill in the art using known methods, such as chemical synthesis or recombinant expression. Other methods for preparing antibodies or antigen-binding antibody fragments are described in WO 2007/070538 (see "Antibodies" on page 22). Those skilled in the art are aware that methods such as so-called phage display libraries (e.g., Morphosys HuCAL Gold) can be compiled and used to discover antibodies or antigen-binding antibody fragments (see WO 2007/070538, pages 24 et seq. and AK Example 1 on page 70 and AK Example 2 on page 72). Other methods for preparing antibodies using DNA libraries derived from B cells are described, for example, on page 26 (WO 2007/070538). Methods for humanizing antibodies are described on pages 30-32 of WO2007070538 and in detail in Queen et al., Pros. Natl. Acad. Sci. USA 86: 10029-10033, 1989 or WO 90/0786. In addition, methods for recombinant expression of proteins in general, and antibodies in particular, are known to those skilled in the art (see, e.g., Berger and Kimrnel (Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152, Academic Press, Inc.); Sambrook et al. (Molecular Cloning: A Laboratory Manual, (Second Edition, Cold Spring Harbor Laboratory Press; Cold Spring Harbor, N.Y.; 1989) Vols. 1-3); Current Protocols in Molecular Biology (F. M. Ausabel et al. [eds.], Current Protocols, Green Publishing Associates, Inc./John Wiley & Sons, Inc.); Harlow et al. (Monoclonal Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988, Paul [ed.]); Fundamental Immunology, (Lippincott Williams & Wilkins (1998)); and Harlow et al. (Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1998)). Those skilled in the art are aware of the corresponding vectors, promoters, and signal peptides necessary for expression of proteins/antibodies. Common methods are also described in WO 2007/070538, pages 41-45. For example, methods for preparing IgG1 antibodies are described in WO 2007/070538, page 74 and in Example 6 thereafter. Methods capable of determining the internalization of an antibody after binding to its antigen are known to those skilled in the art and are described, for example, in WO 2007/070538, page 80. Those skilled in the art can use the methods described in WO 2007/070538, which have been used to prepare carbonic anhydrase IX (Mn) antibodies similarly to the preparation of antibodies specific for other target molecules.

Anti-EGFR antibodies

Examples of antibodies that bind to the cancer target molecule EGFR include cetuximab (INN No. 7906), panitumumab (INN No. 8499), and nimotuzumab (INN No. 8545). Cetuximab (Drug Bank Accession No. DB00002) is a chimeric anti-EGFR1 antibody produced in SP2/0 mouse myeloma cells and sold by ImClone Systems Inc./Merck KgaA/Bristol-Myers Squibb Co. Cetuximab is indicated for the treatment of metastatic, EGFR-expressing colorectal cancers harboring wild-type K-Ras. It has an affinity of ^10-10 M.

sequence:

Cetuximab light chain (κ), SEQ ID NO: 235:

Cetuximab heavy chain, SEQ ID NO: 236:

Panitumumab (INN No. 8499) (Drug Bank Accession No. DB01269) is a recombinant monoclonal human IgG2 antibody that specifically binds to human EGF receptor 1 and is sold by Abgenix/Amgen. Panitumumab was derived from immunization of transgenic (XenoMouse) mice capable of producing human immunoglobulins (light and heavy chains). A specific B cell clone producing anti-EGFR antibodies was selected and immortalized using CHO cells (Chinese Hamster Ovary cells). These cells are now used to produce 100% human antibodies. Panitumumab is indicated for the treatment of EGFR-expressing metastatic colorectal cancer resistant to chemotherapy with fluoropyrimidines, oxaliplatin, and irinotecan. It has an affinity of ^10-11 M.

sequence:

Panitumumab light chain (κ), SEQ ID NO: 237:

Panitumumab heavy chain, SEQ ID NO: 238:

Nimotuzumab (INN No. 8545) (EP 00586002, EP 00712863) is a humanized monoclonal IgG1 antibody that specifically binds to human EGF receptor 1 and is sold by YM BioScienecs Inc. (Mississauga, Canada). It is produced in non-secreting NSO cells (a mammalian cell line). Nimotuzumab is approved for the treatment of head and neck tumors, high-grade astrocytomas and glioblastoma multiforme (not in the EU and US), and pancreatic cancer (Orphan drug, EMA). It has an affinity of ^10⁻ ...

Nimotuzumab light chain, SEQ ID NO: 239:

Nimotuzumab heavy chain, SEQ ID NO: 240:

Further embodiments of EGFR antibodies are as follows:

Zalutumumab/2F8/HuMax-EGFr from Genmab A/S (WO 02/100348, WO 2004/056847, INN No. 8605)

●Necitumumab/11F8, ImClone/IMC-11F8, from ImClone Systems Inc. [EliLilly & Co] (WO 2005/090407 (EP 01735348-A1, US 2007/0264253-A1, US 7,598,350, WO 2005/090407-A1), INN No. 9083)

Matuzumab/anti-EGFR MAb, Merck KGaA/anti-EGFR MAb, Takeda/EMD 72000/EMD-6200/EMD-72000 and EMD-55900/MAb 425/monoclonal antibody 425, from Merck KGaA/Takeda (WO 92/15683, INN No. 8103 (matuzumab))

●RG-7160/GA-201/GA201/R-7160/R7160/RG7160/RO-4858696/RO-5083945/RO4858696/ RO5083945 from Glycart Biotechnology AG (Roche Holding AG) (WO2010/112413-A1, WO 2010/115554)

●GT-MAB 5.2-GEX/CetuGEX from Glycotope GmbH (WO 2008/028686-A2 (EP01900750-A1, EP 01911766-A1, EP 02073842-A2, US 2010/0028947-A1)

ISU-101 from Isu Abxis Inc (ISU Chemical Co Ltd)/Scancell (WO 2008/004834-A1)

ABT-806/mAb-806/ch-806/anti-EGFR monoclonal antibody 806 from Ludwig Institute for Cancer Research/Abbott/Life Science Pharmaceuticals (WO 02/092771, WO 2005/081854, and WO 2009/023265)

SYM-004 (composed of two chimeric IgG1 antibodies (992 and 1024)) from Symphogen A/S (WO2010/022736-A2)

MR1-1/MR1-1KDEL, from IVAX Corp (Teva Pharmaceutical Industries Ltd)(Duke University), (Patent: WO2001/062931-A2)

Antibodies against deletion mutants, EGFRvIII, from Amgen/Abgenix (WO 2005/010151, US 7,628,986)

SC-100 from Scancell Ltd (WO 01/088138-A1)

●MDX-447/EMD 82633/BAB-447/H 447/MAb, EGFR, Medarex/Merck KgaA, from Bristol-Myers Squibb (US)/Merck KGaA (DE)/Takeda (JP), (WO 91/05871, WO 92/15683)

Anti-EGFR-Mab from Xencor (WO 2005/056606)

DXL-1218/anti-EGFR monoclonal antibody (cancer), InNexus, from InNexus Biotechnology Inc, Pharmaprojects PH048638.

In a preferred embodiment, the anti-EGFR antibody is selected from the group consisting of Cetuximab, Panitumumab, Nimotuzumab, Zalutumumab, Necitumumab, Matuzumab, RG-716, GT-MAB 5.2-GEX, ISU-101, ABT-806, SYM-004, MR1-1, SC-100, MDX-447 and DXL-1218.

In a particularly preferred embodiment, the anti-EGFR antibody is selected from the group consisting of Cetuximab, Panitumumab, Nimotuzumab, Zalutumumab, Necitumumab, and Matuzumab.

Those skilled in the art are aware of methods that can be used to prepare other antibodies from the CDR regions of the above antibodies by sequence variation, and these other antibodies have similar or better affinity and/or specificity for the target molecule.

In another embodiment, the anti-EGFR antibody or antigen-binding antibody fragment is selected from

An antibody or antigen-binding antibody fragment comprising three CDR regions of the light chain and three CDR regions of the heavy chain of one of the following antibodies: Cetuximab, Panitumumab, Nimotuzumab, Zalutumumab, Necitumumab, Matuzumab, RG-716, GT-MAB 5.2-GEX, ISU-101, ABT-806, SYM-004, MR1-1, SC-100, MDX-447 and DXL-1218.

In another embodiment, the anti-EGFR antibody or antigen-binding antibody fragment is selected from

An antibody or antigen-binding antibody fragment comprising the three CDR regions of the light chain and the three CDR regions of the heavy chain of one of the following antibodies: Cetuximab, Panitumumab, Nimotuzumab, Zalutumumab, Necitumumab, Matuzumab. These antibodies and their antigen-binding fragments are incorporated herein by reference and can be used in the present invention.

Anti-carbonic anhydrase IX antibody

Examples of antibodies that bind to the cancer target molecule carbonic anhydrase IX are described in WO 2007/070538-A2 (eg, claims 1 - 16).

In a preferred embodiment, the anti-carbonic anhydrase IX antibody or antigen-binding antibody fragment is selected from the group consisting of anti-carbonic anhydrase IX antibody or antigen-binding antibody fragment 3ee9 (claim 4(a) of WO 2007/070538-A2), 3ef2 (claim 4(b) of WO 2007/070538-A2), 1e4 (claim 4(c) of WO 2007/070538-A2), 3a4 (claim 4(d) of WO 2007/070538-A2), 3ab4 (claim 4(e) of WO 2007/070538-A2), 3ah10 (claim 4(f) of WO 2007/070538-A2), 3bb2 (claim 4(g) of WO 2007/070538-A2), 1aa1 (claim 4(h) of WO 2007/070538-A2), and 1a21 (claim 4(i) of WO 2007/070538-A2). (h)), 5a6 (claim 4 (i) of WO 2007/070538-A2) and 5aa3 (claim 4 (j) of WO 2007/070538-A2).

Anti-C4.4a Antibody:

According to the present invention, the C4.4a antibody is used.

Examples of C4.4a antibodies and antigen-binding fragments are described in WO 2012/143499 A2. All antibodies in WO 2012/143499 A2 are incorporated herein by reference into the present specification and can be used in the present invention. The sequences of these antibodies are given in Table 1 of WO 2012/143499 A2, where each row shows the amino acid sequence of the variable light chain or variable heavy chain of the antibody listed in column 1.

In one embodiment, the anti-C4.4a antibody or antigen-binding antibody fragment thereof is internalized by a cell expressing C4.4a after binding to the cell.

In another embodiment, the anti-C4.4a antibody or antigen-binding antibody fragment comprises at least one, two, or three CDR amino acid sequences of an antibody listed in Table 1 of WO 2012/143499 A2 or Table 2 of WO 2012/143499 A2. Preferred embodiments of such antibodies are also listed in WO 2012/143499 A2 and are incorporated herein by reference.

Anti-HER2 antibodies:

An example of an antibody that binds to the cancer target molecule Her2 is trastuzumab (Genentech). Trastuzumab is a humanized antibody used, inter alia, to treat breast cancer.

In addition to trastuzumab (INN 7637, CAS No. RN: 180288-69-1) and pertuzumab (CAS No. 380610-27-5), further examples of antibodies that bind to HER2 are the antibodies disclosed in WO 2009/123894-A2, WO 200/8140603-A2, or WO 2011/044368-A2. An example of an anti-HER2 conjugate is trastuzumab-emtansine (INN No. 9295). These antibodies and antigen-binding fragments thereof are incorporated herein by reference and can be used in the present invention.

Anti-CD20 Antibody:

An example of an antibody that binds to the cancer target molecule CD20 is rituximab (Genentech). Rituximab (CAS No. 174722-31-7) is a chimeric antibody used to treat non-Hodgkin's lymphoma. These antibodies and antigen-binding fragments thereof are incorporated herein by reference and may be used in the present invention.

Anti-CD52 Antibody:

An example of an antibody that binds to the cancer target molecule CD52 is alemtuzumab (Genzyme). Alemtuzumab (CAS No. 216503-57-0) is a humanized antibody used to treat chronic lymphocytic leukemia. These antibodies and antigen-binding fragments thereof are incorporated herein by reference and may be used in the present invention.

Anti-mesothelin antibodies:

Examples of anti-mesothelin antibodies are described, for example, in WO 2009/068204. All antibodies described in WO 2009/068204 are hereby incorporated by reference into the present specification so that these antibodies can be used in the present invention disclosed herein.

Anti-mesothelin antibodies used according to the present invention are also preferably characterized by invariant binding to mesothelin. Invariant binding is characterized, for example, by the antibodies used according to the present invention binding to an epitope of mesothelin that is not masked by other extracellular proteins. Such other extracellular proteins are, for example, the protein ovarian cancer antigen 125 (CA125). Antibodies used are preferably characterized in that CA125 does not block their binding to mesothelin.

Anti-CD30 antibodies

Examples of antibodies that bind to the cancer target molecule CD30 and can be used to treat cancers, such as Hodgkin's lymphoma, include brentuximab, iratumumab, and the antibodies disclosed in WO 2008/092117, WO 2008/036688, or WO 2006/089232. An example of an anti-CD30 conjugate is brentuximab vedotin (INN No. 9144). These antibodies and antigen-binding fragments thereof are incorporated herein by reference and can be used in the present invention.

Anti-CD22 antibodies

Examples of antibodies that bind to the cancer target molecule CD22 and can be used to treat cancers, such as lymphomas, are inotuzumab and epratuzumab. Examples of anti-CD22 conjugates are inotuzumab ozagamycin (INN No. 8574) or anti-CD22-MMAE and anti-CD22-MC-MMAE (CAS RNs: 139504-50-0 and 474645-27-7). These antibodies and their antigen-binding fragments are incorporated herein by reference and can be used in the present invention.

Anti-CD33 antibodies

Examples of antibodies that bind to the cancer target molecule CD33 and can be used to treat cancers, such as leukemia, are gemtuzumab and lintuzumab (INN 7580). An example of an anti-CD33 conjugate is gemtuzumab-ozagamycin. These antibodies and antigen-binding fragments thereof are incorporated herein by reference and can be used in the present invention.

Anti-NMB antibodies

An example of an antibody that binds to the cancer target molecule NMB and can be used to treat cancer, such as melanoma or breast cancer, is Glembatumumab (INN 9199). An example of an anti-NMB conjugate is Glembatumumab Vedotin (CASRN: 474645-27-7). These antibodies and antigen-binding fragments thereof are incorporated herein by reference and can be used in the present invention.

Anti-CD56 antibody

An example of an antibody that binds to the cancer target molecule CD56 and can be used to treat cancers such as multiple myeloma, small cell lung cancer, MCC, or ovarian cancer is Lorvotuzumab. An example of an anti-CD56 conjugate is Lorvotuzumab Mertansine (CAS RN: 139504-50-0). These antibodies and antigen-binding fragments thereof are incorporated herein by reference and can be used in the present invention.

Anti-CD70 antibodies

Examples of antibodies that bind to the cancer target molecule CD70 and can be used to treat cancers, such as non-Hodgkin's lymphoma or renal cell carcinoma, are disclosed in WO 2007/038637-A2 and WO 2008/070593-A2. An example of an anti-CD70 conjugate is SGN-75 (CD70 MMAF). These antibodies and antigen-binding fragments thereof are incorporated herein by reference and can be used in the present invention.

Anti-CD74 antibody

An example of an antibody that binds to the cancer target molecule CD74 and can be used to treat cancer, such as multiple myeloma, is Milatuzumab. An example of an anti-CD74 conjugate is Milatuzumab-doxorubicin (CAS RN: 23214-92-8). These antibodies and antigen-binding fragments thereof are incorporated herein by reference and can be used in the present invention.

Anti-CD19 antibodies

An example of an antibody that binds to the cancer target molecule CD19 and can be used to treat cancer, such as non-Hodgkin's lymphoma, is disclosed in WO 2008/031056-A2. Examples of other antibodies and anti-CD19 conjugates are disclosed in WO 2008/047242-A2. These antibodies and antigen-binding fragments thereof are incorporated herein by reference, and they can be used in the present invention.

Anti-mucin antibodies

Examples of antibodies that bind to the cancer target molecule mucin-1 and can be used to treat cancers, such as non-Hodgkin's lymphoma, are clivatuzumab or the antibodies disclosed in WO 2003/106495-A2 and WO 2008/028686-A2. Examples of anti-mucin conjugates are disclosed in WO 2005/009369-A2. These antibodies and antigen-binding fragments thereof are incorporated herein by reference and can be used in the present invention.

Anti-CD138 antibody

Antibodies and conjugates thereof that bind to the cancer target molecule CD138 can be used to treat cancer, for example, examples of multiple myeloma are disclosed in WO 2009/080829-A1 and WO 2009/080830-A1. These antibodies and antigen-binding fragments thereof are incorporated herein by reference and can be used in the present invention.

Anti-integrin-αV antibodies

Examples of antibodies that bind to the cancer target molecule integrin αV and can be used to treat cancers, such as melanoma, sarcoma, or carcinoma, include intetumumab (CAS RN: 725735-28-4), abciximab (CAS RN: 143653-53-6), etaracizumab (CAS RN: 892553-42-3), or antibodies disclosed in US Pat. No. 7,465,449, EP 719859-A1, WO 2002/012501-A1, or WO 2006/062779-A2. Examples of anti-integrin αV conjugates include intetumumab-DM4 and other ADCs disclosed in WO 2007/024536-A2. These antibodies and antigen-binding fragments thereof are incorporated herein by reference and can be used in the present invention.

Anti-TDGF1 antibody

Examples of antibodies that bind to the cancer target molecule TDGF1 and can be used to treat cancer are those disclosed in WO 02/077033-A1, US Pat. No. 7,318,924, WO 2003/083041-A2, and WO 2002/088170-A2. Examples of anti-TDGF1 conjugates are disclosed in WO 2002/088170-A2. These antibodies and antigen-binding fragments thereof are incorporated herein by reference and can be used in the present invention.

Anti-PSMA antibodies

Examples of antibodies that bind to the cancer target molecule PSMA and can be used to treat cancer, such as prostate cancer, are those disclosed in WO 97/35616-A1, WO 99/47554-A1, WO 01/009192-A1, and WO 2003/034903. Examples of anti-PSMA conjugates are disclosed in WO 2009/026274-A1 and WO 2007/002222. These antibodies and antigen-binding fragments thereof are incorporated herein by reference and can be used in the present invention.

Anti-EPHA2 antibody

Examples of antibodies that bind to the cancer target molecule EPHA2 and can be used to prepare conjugates and for treating cancer are disclosed in WO 2004/091375-A2. These antibodies and antigen-binding fragments thereof are incorporated herein by reference and can be used in the present invention.

Anti-SLC44A4 antibody

Examples of antibodies that bind to the cancer target molecule SLC44A4 and can be used to prepare conjugates and treat cancers, such as pancreatic or prostate cancer, are disclosed in WO2009/033094-A2 and US2009/0175796-A1. These antibodies and antigen-binding fragments thereof are incorporated herein by reference and can be used in the present invention.

anti-HLA-DOB antibodies

An example of an antibody that binds to the cancer target molecule HLA-DOB is the antibody Lym-1 (CAS RN: 301344-99-0), which can be used to treat cancers such as non-Hodgkin's lymphoma. Examples of anti-HLA-DOB conjugates are disclosed, for example, in WO2005/081711-A2. These antibodies and antigen-binding fragments thereof are incorporated herein by reference and can be used in the present invention.

Anti-VTCN1 antibodies

Examples of antibodies that bind to the cancer target molecule VTCN1 and can be used to prepare conjugates and treat cancers, such as ovarian cancer, pancreatic cancer, lung cancer, or breast cancer, are disclosed in WO 2006/074418-A2. These antibodies and antigen-binding fragments thereof are incorporated herein by reference and can be used in the present invention.

Anti-FGFR2 antibodies

According to the present invention, anti-FGFR2 antibodies may be used.

Examples of anti-FGFR2 antibodies and antigen-binding fragments are described in WO2013076186. All antibodies of WO2013076186 are incorporated herein by reference into the present specification and can be used in the present invention. The sequences of these antibodies are shown in Tables 9 and 10 of WO2013076186. Preferred are antibodies, antigen-binding fragments, and antibody variants derived from antibodies designated M048-D01 and M047-D08. Preferred anti-FGFR2 antibodies bind to various splice variants of FGFR2.

In one embodiment, the anti-FGFR2 antibody or antigen-binding antibody fragment thereof is internalized by a cell expressing FGFR2 upon binding to the cell.

In another embodiment, the anti-FGFR2 antibody or antigen-binding antibody fragment comprises at least one, two or three CDR amino acid sequences of an antibody listed in Table 9 or Table 10 of WO2013076186. Preferred embodiments of such antibodies are also listed in WO2013076186 and are incorporated herein by reference.

Anti-TWEAKR antibody

In a preferred embodiment, when an anti-TWEAKR antibody or antigen-binding fragment thereof is used in the method of the present invention, the one or more antibodies or fragments are selected from those described below. In addition, antibodies that bind to TWEAKR are known to those skilled in the art, see, for example, WO2009/020933 (A2) or WO2009140177 (A2).

The present invention particularly relates to conjugates containing antibodies or antigen-binding antibody fragments or variants thereof that strongly activate TWEAKR (SEQ ID NO: 169 (protein); SEQ ID NO: 170 (DNA)), thereby strongly inducing apoptosis in various cancer cells that overexpress TWEAKR.

The described anti-TWEAKR antibodies (e.g., PDL-192) have limited TWEAKR agonist activity in inducing apoptosis and inhibiting proliferation and do not approach the potency of the endogenous ligand, TWEAK. This lack of agonist activity is not based on reduced affinity, as these antibodies bind to TWEAKR with affinities in a similar range compared to the endogenous ligand, TWEAK (Michaelson JS et al., MAbs. 2011 Jul-Aug;3(4):362-75; Culp PA et al., Clin Cancer Res. 2010 Jan 15;16(2):497-508), and even antibodies with higher binding affinity do not necessarily exhibit more potent signaling activity (Culp PA et al., Clin Cancer Res. 2010 Jan 15;16(2):497-508). Furthermore, it has been shown that the anti-tumor activity of the described antibodies is dependent on Fc effector function and that ADCC plays an important role in in vivo efficacy in mouse models.

Generation of anti-TWEAKR antibodies

A complete human antibody phage library (Hoet RM et al., Nat Biotechnol 2005; 23(3): 344-8) was used to isolate the TWEAKR-specific human monoclonal antibodies of the present invention (Hoogenboom H.R., Nat Biotechnol 2005; 23(3): 1105-16) by protein screening (panning) using the dimeric Fc-fused extracellular domains of human and mouse TWEAKR as immobilized targets. Eleven different Fab phages were identified, and the corresponding antibodies were cloned into a mammalian IgG expression vector that provides the CH2-CH3 domain deleted in soluble Fabs. After the preferred antibodies were identified, these were expressed as full-length IgG. These constructs were transiently expressed, for example, in mammalian cells as described in Tom et al., Methods Express, Chapter 12: Expression Systems, edited by Michael R. Dyson and Yves Durocher, Scion Publishing Ltd, 2007 (see AK-Example 1). The antibodies were purified by protein-A chromatography and further characterized by their binding affinity to soluble monomeric TWEAKR using ELISA and BIAcore analysis as described in AK-Example 2. To determine the cellular binding characteristics of the anti-TWEAKR antibodies, binding was tested by flow cytometry on a number of cell lines (HT29, HS68, HS578). An NF-κB reporter gene assay was performed to examine the agonistic activity of all 11 identified antibodies (human IgG1). The antibody with the highest in vitro potency (TPP-883) was selected for further potency and affinity maturation (see AK-Example 1 for details). A single substitution variant with improved agonistic activity was tested: G102T in CDR-H3. Finally, seven variants were selected based on their improved affinity compared to the best single substitution variant, G102T. The corresponding DNA was cloned into a mammalian IgG expression vector and functional activity was tested in the NF-κB reporter gene assay described above. Finally, the resulting sequences were compared to the human germline sequences, and deviations that did not significantly affect affinity and potency were corrected. The following antibodies were obtained by antibody library screening and by affinity and/or potency maturation: "TPP-2090", "TPP-2149", "TPP-2093", "TPP-2148", "TPP-2084", "TPP-2077", "TPP-1538", "TPP-883", "TPP-1854", "TPP-1853", "TPP-1857" and "TPP-1858".

Antibodies of the present invention can also be obtained by methods known in the art, such as antibody phage display screening (see, e.g., Hoet RM et al., Nat Biotechnol 2005; 23(3):344-8), established hybridoma technology (see, e.g., Köhler and Milstein Nature. 1975 Aug 7; 256(5517):495-7), or immunization of mice, in particular immunization of hMAb mice (e.g., VelocImmune mouse®).

Specific embodiments of anti-TWEAKR antibodies

One embodiment of the present invention is to provide an antibody or antigen-binding antibody fragment or variant thereof that exhibits strong induction of caspase 3/7 in one or more TWEAKR-expressing cell lines. In a preferred embodiment, the one or more TWEAKR-expressing cell lines are selected from the group consisting of WiDr, A253, NCI-H322, HT29, and 786-O. "Induction of caspase 3/7" can be measured by conventional methods known in the art, including those described herein. In one embodiment, "induction of caspase 3/7" is measured according to the present invention by measuring activity with a caspase 3/7 solution (Promega, #G8093) and reading luminescence on a VICTOR V (Perkin Elmer). At the end of the incubation period, caspase 3/7 activity is measured and the induction coefficient of caspase 3/7 is determined by comparison with untreated cells. An antibody is described as exhibiting "strong induction" of caspase 3/7 when the induction coefficient is greater than 1.2, preferably greater than 1.5, even more preferably greater than 1.8, even more preferably greater than 2.1, and even more preferably greater than 2.5. Anti-TWEAKR antibodies are provided that result in stronger induction of caspase 3/7 in HT29 cells than described agonistic antibodies [e.g., PDL-192 (TPP-1104), P4A8 (TPP-1324), 136.1 (TPP-2194)] and compared to 300 ng/ml recombinant human TWEAK. This potent induction of caspase 3/7 in cancer cells was also observed in WiDr, A253, NIC-H322, and 786-O cells, with the antibodies of the invention tested eliciting higher coefficients of variation in most experiments compared to the reference antibodies [PDL-192 (TPP-1104), P4A8 (TPP-1324)] and 300 ng/ml TWEAK. Some of the antibodies of the invention bind to TWEAKR with only moderate affinities (>10 nM) that are significantly lower than that of the endogenous ligand TWEAK and also lower than that of other known agonistic antibodies. This property offers further potential advantages, such as the potential for deeper tumor penetration.

In this regard, one embodiment of the present invention provides antibodies or antigen-binding antibody fragments thereof that specifically bind to a novel epitope on TWEAKR, characterized by selective binding to the aspartic acid (D) at position 47 (D47) of TWEAKR (SEQ ID NO: 169; see also Figure 1). The reliance on recognition of certain TWEAKR amino acids for antibody interaction correlates with the agonistic activity measured for these antibodies. The natural ligand, TWEAK, exhibits potent activation of TWEAKR and is dependent on binding to leucine 46 within the cysteine-rich domain of TWEAKR (Pellegrini et al., FEBS 280:1818-1829). P4A8 exhibits minimal agonistic activity and interacts at least partially with domains outside the cysteine-rich domain of TWEAKR. PDL-192 exhibits moderate agonistic activity and is dependent on binding to R56 within the cysteine-rich domain, opposite the TWEAK ligand site. Antibodies of the present invention (e.g., TPP-2090) are dependent on D47 for binding, while TWEAK is dependent on L46 for binding. Thus, TWEAK binds to a similar but distinct binding site ( FIG. 7 ). Thus, the antibodies of the present invention that exhibit strong agonistic activity bind to a novel epitope of the antibody associated with very high agonistic activity (D47-dependent).

The amino acid at position 47 (D47) of TWEAKR (SEQ ID NO: 169) is considered to be critical for the binding of the antibody according to the present invention, which means that when the antibody loses more than 20%, or more than 30%, or more than 40%, or more than 50%, or more than 60%, or more than 70%, or more than 80%, or more than 90%, or 100% of its ELISA signal by changing its residue to alanine as described in AK-Example 2 and Figure 6, the antibody specifically binds to D at position 47 (D47) of TWEAKR (SEQ ID NO: 169). Alternatively, the antibody specifically binds to D at position 47 (D47) of TWEAKR (SEQ ID NO: 169) when the antibody loses more than 20%, or more than 30%, or more than 40%, or more than 50%, or more than 60%, or more than 70%, or more than 80%, or more than 90%, or 100% of its ELISA signal for TPP-2614 as compared to TPP-2203. Preferably, the antibody specifically binds to D at position 47 (D47) of TWEAKR (SEQ ID NO: 169) when the antibody loses more than 80% of its ELISA signal for TPP-2614 as compared to TPP-2203.

In this application, reference is made to the following preferred antibodies of the present invention as shown in the following table: "TPP-2090", "TPP-2149", "TPP-2093", "TPP-2148", "TPP-2084", "TPP-2077", "TPP-1538", "TPP-883", "TPP-1854", "TPP-1853", "TPP-1857", "TPP-1858".

TPP-2090 is an antibody comprising a heavy chain region corresponding to SEQ ID NO: 2 and a light chain region corresponding to SEQ ID NO: 1.

TPP-2149 is an antibody comprising a heavy chain region corresponding to SEQ ID NO: 12 and a light chain region corresponding to SEQ ID NO: 11.

TPP-2093 is an antibody comprising a heavy chain region corresponding to SEQ ID NO: 22 and a light chain region corresponding to SEQ ID NO: 21.

TPP-2148 is an antibody comprising a heavy chain region corresponding to SEQ ID NO: 32 and a light chain region corresponding to SEQ ID NO: 31.

TPP-2084 is an antibody comprising a heavy chain region corresponding to SEQ ID NO: 42 and a light chain region corresponding to SEQ ID NO: 41.

TPP-2077 is an antibody comprising a heavy chain region corresponding to SEQ ID NO: 52 and a light chain region corresponding to SEQ ID NO: 51.

TPP-1538 is an antibody comprising a heavy chain region corresponding to SEQ ID NO: 62 and a light chain region corresponding to SEQ ID NO: 61.

TPP-883 is an antibody comprising a heavy chain region corresponding to SEQ ID NO: 72 and a light chain region corresponding to SEQ ID NO: 71.

TPP-1854 is an antibody comprising a heavy chain region corresponding to SEQ ID NO: 82 and a light chain region corresponding to SEQ ID NO: 81.

TPP-1853 is an antibody comprising a heavy chain region corresponding to SEQ ID NO: 92 and a light chain region corresponding to SEQ ID NO: 91.

TPP-1857 is an antibody comprising a heavy chain region corresponding to SEQ ID NO: 102 and a light chain region corresponding to SEQ ID NO: 101.

TPP-1858 is an antibody comprising a heavy chain region corresponding to SEQ ID NO: 112 and a light chain region corresponding to SEQ ID NO: 111.

TPP-2090 is an antibody comprising a heavy chain variable region corresponding to SEQ ID NO: 10 and a light chain variable region corresponding to SEQ ID NO: 9.

TPP-2149 is an antibody comprising a heavy chain variable region corresponding to SEQ ID NO: 20 and a light chain variable region corresponding to SEQ ID NO: 19.

TPP-2093 is an antibody comprising a heavy chain variable region corresponding to SEQ ID NO: 30 and a light chain variable region corresponding to SEQ ID NO: 29.

TPP-2148 is an antibody comprising a heavy chain variable region corresponding to SEQ ID NO: 40 and a light chain variable region corresponding to SEQ ID NO: 39.

TPP-2084 is an antibody comprising a heavy chain variable region corresponding to SEQ ID NO: 50 and a light chain variable region corresponding to SEQ ID NO: 49.

TPP-2077 is an antibody comprising a heavy chain variable region corresponding to SEQ ID NO: 60 and a light chain variable region corresponding to SEQ ID NO: 59.

TPP-1538 is an antibody comprising a heavy chain variable region corresponding to SEQ ID NO: 70 and a light chain variable region corresponding to SEQ ID NO: 69.

TPP-883 is an antibody comprising a heavy chain variable region corresponding to SEQ ID NO: 80 and a light chain variable region corresponding to SEQ ID NO: 79.

TPP-1854 is an antibody comprising a heavy chain variable region corresponding to SEQ ID NO: 90 and a light chain variable region corresponding to SEQ ID NO: 89.

TPP-1853 is an antibody comprising a heavy chain variable region corresponding to SEQ ID NO: 100 and a light chain variable region corresponding to SEQ ID NO: 99.

TPP-1857 is an antibody comprising a heavy chain variable region corresponding to SEQ ID NO: 110 and a light chain variable region corresponding to SEQ ID NO: 109.

TPP-1858 is an antibody comprising a heavy chain variable region corresponding to SEQ ID NO: 120 and a light chain variable region corresponding to SEQ ID NO: 119.

Preferred embodiments of anti-TWEAKR antibodies are those that are:

1. An anti-TWEAKR antibody or an antigen-binding fragment thereof that specifically binds to D at position 47 (D47) of TWEAKR (SEQ ID NO: 169).

2. The antibody or antigen-binding fragment thereof according to embodiment 1, wherein the antibody is an agonistic antibody.

3. The antibody or antigen-binding fragment thereof according to embodiment 1 or 2, comprising:

A variable heavy chain comprising:

(a) a heavy chain CDR1 encoded by an amino acid sequence comprising the formula PYPMX (SEQ ID NO: 171), wherein X is I or M;

(b) a CDR2 of the heavy chain encoded by an amino acid sequence comprising the formula YISPSGGXTHYADSVKG (SEQ ID NO: 172), wherein X is S or K; and

(c) CDR3 of the heavy chain encoded by an amino acid sequence comprising the formula GGDTYFDYFDY (SEQ ID NO: 173);

and a variable light chain comprising:

(a) a CDR1 of a light chain encoded by an amino acid sequence comprising the formula RASQSISXYLN (SEQ ID NO: 174), wherein X is G or S;

(b) a CDR2 of the light chain encoded by an amino acid sequence comprising the formula XASSLQS (SEQ ID NO: 175), wherein X is Q, A or N; and

(c) a CDR3 of the light chain encoded by an amino acid sequence comprising the formula QQSYXXPXIT (SEQ ID NO: 176), wherein X at position 5 is T or S, X at position 6 is T or S, and X at position 8 is G or F.

4. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, comprising:

a. a variable heavy chain comprising a heavy chain variable CDR1 sequence as shown in SEQ ID NO: 6, a heavy chain variable CDR2 sequence as shown in SEQ ID NO: 7, and a heavy chain variable CDR3 sequence as shown in SEQ ID NO: 8, and

A variable light chain comprising a light chain variable CDR1 sequence as shown in SEQ ID NO: 3, a light chain variable CDR2 sequence as shown in SEQ ID NO: 4, and a light chain variable CDR3 sequence as shown in SEQ ID NO: 5; or

b. a variable heavy chain comprising a heavy chain variable CDR1 sequence as shown in SEQ ID NO: 16, a heavy chain variable CDR2 sequence as shown in SEQ ID NO: 17, a heavy chain variable CDR3 sequence as shown in SEQ ID NO: 18, and

A variable light chain comprising a light chain variable CDR1 sequence as shown in SEQ ID NO: 13, a light chain variable CDR2 sequence as shown in SEQ ID NO: 14, and a light chain variable CDR3 sequence as shown in SEQ ID NO: 15; or

c. a variable heavy chain comprising a heavy chain variable CDR1 sequence as shown in SEQ ID NO: 26, a heavy chain variable CDR2 sequence as shown in SEQ ID NO: 27, a heavy chain variable CDR3 sequence as shown in SEQ ID NO: 28, and

A variable light chain comprising a light chain variable CDR1 sequence as shown in SEQ ID NO: 23, a light chain variable CDR2 sequence as shown in SEQ ID NO: 24, and a light chain variable CDR3 sequence as shown in SEQ ID NO: 25; or

d. a variable heavy chain comprising a heavy chain variable CDR1 sequence as shown in SEQ ID NO: 36, a heavy chain variable CDR2 sequence as shown in SEQ ID NO: 37, a heavy chain variable CDR3 sequence as shown in SEQ ID NO: 38, and

A variable light chain comprising a light chain variable CDR1 sequence as shown in SEQ ID NO: 33, a light chain variable CDR2 sequence as shown in SEQ ID NO: 34, and a light chain variable CDR3 sequence as shown in SEQ ID NO: 35; or

e. a variable heavy chain comprising a heavy chain variable CDR1 sequence as shown in SEQ ID NO: 46, a heavy chain variable CDR2 sequence as shown in SEQ ID NO: 47, a heavy chain variable CDR3 sequence as shown in SEQ ID NO: 48, and

A variable light chain comprising a light chain variable CDR1 sequence as shown in SEQ ID NO: 43, a light chain variable CDR2 sequence as shown in SEQ ID NO: 44, and a light chain variable CDR3 sequence as shown in SEQ ID NO: 45; or

f. a variable heavy chain comprising a heavy chain variable CDR1 sequence as shown in SEQ ID NO: 56, a heavy chain variable CDR2 sequence as shown in SEQ ID NO: 57, a heavy chain variable CDR3 sequence as shown in SEQ ID NO: 58, and

A variable light chain comprising a light chain variable CDR1 sequence as shown in SEQ ID NO: 53, a light chain variable CDR2 sequence as shown in SEQ ID NO: 54, and a light chain variable CDR3 sequence as shown in SEQ ID NO: 55; or

g. a variable heavy chain comprising a heavy chain variable CDR1 sequence as shown in SEQ ID NO: 66, a heavy chain variable CDR2 sequence as shown in SEQ ID NO: 67, a heavy chain variable CDR3 sequence as shown in SEQ ID NO: 68, and

A variable light chain comprising a light chain variable CDR1 sequence as shown in SEQ ID NO: 63, a light chain variable CDR2 sequence as shown in SEQ ID NO: 64, and a light chain variable CDR3 sequence as shown in SEQ ID NO: 65; or

h. a variable heavy chain comprising a heavy chain variable CDR1 sequence as shown in SEQ ID NO: 76, a heavy chain variable CDR2 sequence as shown in SEQ ID NO: 77, a heavy chain variable CDR3 sequence as shown in SEQ ID NO: 78, and

A variable light chain comprising a light chain variable CDR1 sequence as shown in SEQ ID NO: 73, a light chain variable CDR2 sequence as shown in SEQ ID NO: 74, and a light chain variable CDR3 sequence as shown in SEQ ID NO: 75; or

i. a variable heavy chain comprising a heavy chain variable CDR1 sequence as shown in SEQ ID NO: 86, a heavy chain variable CDR2 sequence as shown in SEQ ID NO: 87, a heavy chain variable CDR3 sequence as shown in SEQ ID NO: 88, and

A variable light chain comprising a light chain variable CDR1 sequence as shown in SEQ ID NO: 83, a light chain variable CDR2 sequence as shown in SEQ ID NO: 84, and a light chain variable CDR3 sequence as shown in SEQ ID NO: 85; or

j. a variable heavy chain comprising a heavy chain variable CDR1 sequence as shown in SEQ ID NO: 96, a heavy chain variable CDR2 sequence as shown in SEQ ID NO: 97, a heavy chain variable CDR3 sequence as shown in SEQ ID NO: 98, and

A variable light chain comprising a light chain variable CDR1 sequence as shown in SEQ ID NO: 93, a light chain variable CDR2 sequence as shown in SEQ ID NO: 94, and a light chain variable CDR3 sequence as shown in SEQ ID NO: 95; or

k. a variable heavy chain comprising a heavy chain variable CDR1 sequence as shown in SEQ ID NO: 106, a heavy chain variable CDR2 sequence as shown in SEQ ID NO: 107, a heavy chain variable CDR3 sequence as shown in SEQ ID NO: 108, and

A variable light chain comprising a light chain variable CDR1 sequence as shown in SEQ ID NO: 103, a light chain variable CDR2 sequence as shown in SEQ ID NO: 104, and a light chain variable CDR3 sequence as shown in SEQ ID NO: 105; or

1. A variable heavy chain comprising a heavy chain variable CDR1 sequence as shown in SEQ ID NO: 116, a heavy chain variable CDR2 sequence as shown in SEQ ID NO: 117, a heavy chain variable CDR3 sequence as shown in SEQ ID NO: 118, and

A variable light chain comprising the light chain variable CDR1 sequence as shown in SEQ ID NO: 113, the light chain variable CDR2 sequence as shown in SEQ ID NO: 114, and the light chain variable CDR3 sequence as shown in SEQ ID NO: 115.

5. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, comprising:

a. a variable sequence of the heavy chain as shown in SEQ ID NO: 10 and a variable sequence of the light chain as shown in SEQ ID NO: 9, or

b. the variable sequence of the heavy chain as shown in SEQ ID NO: 20 and the variable sequence of the light chain as shown in SEQ ID NO: 19, or

c. the variable sequence of the heavy chain as shown in SEQ ID NO: 30 and the variable sequence of the light chain as shown in SEQ ID NO: 29, or

d. a heavy chain variable sequence as shown in SEQ ID NO: 40 and a light chain variable sequence as shown in SEQ ID NO: 39, or

e. the variable sequence of the heavy chain as shown in SEQ ID NO: 50 and the variable sequence of the light chain as shown in SEQ ID NO: 49, or

f. the variable sequence of the heavy chain as shown in SEQ ID NO: 60 and the variable sequence of the light chain as shown in SEQ ID NO: 59, or

g. the variable sequence of the heavy chain as shown in SEQ ID NO: 70 and the variable sequence of the light chain as shown in SEQ ID NO: 69, or

h. the variable sequence of the heavy chain as shown in SEQ ID NO: 80 and the variable sequence of the light chain as shown in SEQ ID NO: 79, or

i. the variable sequence of the heavy chain as shown in SEQ ID NO: 90 and the variable sequence of the light chain as shown in SEQ ID NO: 89, or

j. the variable sequence of the heavy chain as shown in SEQ ID NO: 100 and the variable sequence of the light chain as shown in SEQ ID NO: 99, or

k. the variable sequence of the heavy chain as shown in SEQ ID NO: 110 and the variable sequence of the light chain as shown in SEQ ID NO: 109, or

1. The variable sequence of the heavy chain is shown in SEQ ID NO: 120 and the variable sequence of the light chain is shown in SEQ ID NO: 119.

6. The antibody according to any one of the preceding embodiments, which is an IgG antibody.

7. The antibody according to any one of the preceding embodiments, comprising:

a. The heavy chain sequence as shown in SEQ ID NO: 2 and the light chain sequence as shown in SEQ ID NO: 1, or

b. the sequence of the heavy chain as shown in SEQ ID NO: 12 and the sequence of the light chain as shown in SEQ ID NO: 11, or

c. the heavy chain sequence as shown in SEQ ID NO: 22 and the light chain sequence as shown in SEQ ID NO: 21, or

d. the heavy chain sequence as shown in SEQ ID NO: 32 and the light chain sequence as shown in SEQ ID NO: 31, or

e. the sequence of the heavy chain as shown in SEQ ID NO: 42 and the sequence of the light chain as shown in SEQ ID NO: 41, or

f. the heavy chain sequence as shown in SEQ ID NO: 52 and the light chain sequence as shown in SEQ ID NO: 51, or

g. the heavy chain sequence as shown in SEQ ID NO: 62 and the light chain sequence as shown in SEQ ID NO: 61, or

h. the sequence of the heavy chain as shown in SEQ ID NO: 72 and the sequence of the light chain as shown in SEQ ID NO: 71, or

i. the sequence of the heavy chain as shown in SEQ ID NO: 82 and the sequence of the light chain as shown in SEQ ID NO: 81, or

j. the sequence of the heavy chain as shown in SEQ ID NO: 92 and the sequence of the light chain as shown in SEQ ID NO: 91, or

k. the sequence of the heavy chain as shown in SEQ ID NO: 102 and the sequence of the light chain as shown in SEQ ID NO: 101, or

1. The sequence of the heavy chain is shown in SEQ ID NO: 112 and the sequence of the light chain is shown in SEQ ID NO: 111.

8. The antigen-binding fragment according to any one of the preceding embodiments or the antigen-binding fragment of the antibody according to any one of the preceding embodiments, which is a scFv, Fab, Fab ̓ fragment or F(ab ̓)2 fragment.

9. The antibody or antigen-binding fragment according to any one of the preceding embodiments, which is a monoclonal antibody or antigen-binding fragment thereof.

10. The antibody or antigen-binding fragment according to any one of the preceding embodiments, which is a human, humanized or chimeric antibody or antigen-binding fragment.

Particularly preferred is the anti-TWEAKR antibody TPP-2090.

Isotopes, salts, solvates, isotopic variants

The present invention also includes all suitable isotopic variants of the compounds of the present invention. Isotopic variants of the compounds of the present invention are understood herein to mean compounds in which at least one atom in the compounds of the present invention has been replaced by another atom having the same atomic number but an atomic mass different from the atomic mass usually or predominantly present in nature. Examples of isotopes that can be incorporated into the compounds of the present invention are isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, bromine, and iodine, such as ² H (deuterium), ³ H (tritium), ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁷ O, ¹⁸ O, ³² P, ³³ P, ³³ S, ³⁴ S, ³⁵ S, ³⁶ S, ¹⁸ F, ³⁶ Cl, ⁸² Br, ¹²³ I, ¹²⁴ I, ¹²⁹ I, and ¹³¹ I. Certain isotopic variants of the compounds of the present invention, especially those into which one or more radioactive isotopes have been incorporated, may be useful, for example, for examining the mechanism of action or the distribution of the active substance in vivo; compounds labeled with ³ H or ¹⁴ C isotopes are particularly suitable for this purpose due to their relative ease of preparation and detection. In addition, the incorporation of isotopes, such as deuterium, may confer specific therapeutic benefits due to the greater metabolic stability of the compound, such as an increase in half-life in vivo or a reduction in the required active dose; such modifications of the compounds of the present invention therefore optionally also constitute a preferred embodiment of the present invention. Isotopic variants of the compounds of the present invention can be prepared by methods known to those skilled in the art, for example, by the procedures described in the following methods and examples, by using the respective reagents and/or corresponding isotopic modifications of the starting compounds.

Preferred salts in the present invention are physiologically acceptable salts of the compounds according to the invention. Salts which are not suitable for pharmaceutical uses themselves but which can be used, for example, for the isolation or purification of the compounds according to the invention are also included.

Physiologically acceptable salts of the compounds of the present invention include acid addition salts of inorganic acids, carboxylic acids and sulfonic acids, for example, salts of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalene disulfonic acid, acetic acid, trifluoroacetic acid, propionic acid, lactic acid, tartaric acid, malic acid, citric acid, fumaric acid, maleic acid and benzoic acid.

Physiologically acceptable salts of the compounds according to the invention also include salts with customary bases, such as and preferably alkali metal salts (e.g. sodium and potassium salts), alkaline earth metal salts (e.g. calcium and magnesium salts) and ammonium salts derived from ammonia or organic amines having 1 to 16 carbon atoms, such as and preferably ethylamine, diethylamine, triethylamine, ethyldiisopropylamine, monoethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, dimethylaminoethanol, procaine, dibenzylamine, N -methylpiperidine, N-methylmorpholine, arginine, lysine and 1,2-ethylenediamine.

Solvates are described herein as those forms of the compounds of the invention which form complexes in the solid or liquid state by coordination with solvent molecules. Hydrates are a specific form of solvates in which coordination occurs with water. Preferred solvates in the present invention are hydrates.

Furthermore, the present invention also includes prodrugs of the compounds of the present invention. The term "prodrug" herein refers to a compound that is biologically active or inactive itself but is converted into a compound of the present invention during its residence time in the body (e.g., by metabolism or hydrolysis).

Specific implementation plans

The following embodiments are particularly preferred:

Implementation A:

ADC of the following formula and its salt, solvate and solvate salt

wherein KSP-L- is a compound of the following formula (II), (IIa), (IIb), (IIc), (IId), (IIe) or the following formula (IIf), the binding entity is an anti-TWEAKR antibody (particularly preferably an anti-TWEAKR antibody that specifically binds to amino acid D (D47) at position 47 of TWEAKR (SEQ ID NO: 169), in particular the anti-TWEAKR antibody TPP-2090), and n is a value from 1 to 10:

Formula (IIf):

in

_X1 represents N, _X2 represents N and _X3 represents C;

_X1 represents CH, _X2 represents C and _X3 represents N;

_X1 represents NH, _X2 represents C and _X3 represents C; or

_X1 represents CH, _X2 represents N and _X3 represents C;

A represents CO (carbonyl);

R ¹ represents –L-#1, H, –COOH, –CONHNH ₂ , –(CH ₂ ) _1-3 NH ₂ , –CONZ′′(CH ₂ ) _1-3 NH ₂ and –CONZ′′CH ₂ COOH, wherein Z′′ represents H or NH ₂ ;

R ² and R ⁴ represent H or –L-#1, or R ² and R ⁴ together (forming a pyrrolidine ring) represent -CH ₂ -CHR ¹⁰ - or –CHR ¹⁰ -CH ₂ -, wherein R ¹⁰ represents H or –L-#1;

R ³ represents –L-#1 or C _1-10 -alkyl-, which may be optionally substituted by –OH, O-alkyl, SH, S-alkyl, O-CO-alkyl, O-CO-NH-alkyl, NH-CO-alkyl, NH-CO-NH-alkyl, S(O) _n -alkyl, SO ₂ -NH-alkyl, NH-alkyl, N(alkyl) ₂ or NH ₂ (wherein alkyl is preferably C _1-3 -alkyl);

R ⁵ represents –L-#1, H or F;

R ⁶ and R ⁷ independently of one another represent H, (optionally fluorinated) C _1-3 -alkyl, (optionally fluorinated) C _2-4 -alkenyl, (optionally fluorinated) C _2-4 -alkynyl, hydroxy or halogen;

R ⁸ represents a branched C _1-5 -alkyl group which may be substituted with -L-#1; and

^R9 represents H or F,

wherein one of the substituents R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁸ and R ¹⁰ represents -L-#1, and

–L- represents the linker and #1 represents the bond to the antibody.

The linker is preferably a linker

§-(CO)m-L1-L2-§§

in

m is 0 or 1;

§ represents the key to KSP and

§§ represents the bond to the antibody, and

L2 Representative

in

# ¹ refers to the connection point with the sulfur atom of the antibody,

# ² refers to the connection point with the group L ¹ ,

And L1 is expressed by the following formula

#1–(NR ¹⁰ ) _n -(G1) _o -G2- ^#2

in

R ¹⁰ represents H, NH ₂ or C ₁ -C ₃ -alkyl;

G1 stands for –NHCO- or;

n is 0 or 1;

o is 0 or 1; and

G2 represents a linear or branched hydrocarbon chain having 1 to 100 carbon atoms, which is derived from arylene and/or linear and/or branched and/or cyclic alkylene and may be interrupted one or more times by one or more of the radicals -O-, -S-, -SO-, SO ₂ , -NH-, -CO-, -NHCO-, -CONH-, -NMe-, -NHNH-, -SO ₂ NHNH-, -CONHNH- and a 3- to 10-membered aromatic or nonaromatic heterocycle (preferably) having up to 4 heteroatoms selected from N, O and S or -SO-, where side chains, if present, may be substituted by -NHCONH ₂ , -COOH, -OH, -NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid.

Here, # ¹ is the bond to the KSP inhibitor and # ² is the bond to the coupling group (e.g., L2) of the binder.

Implementation Plan B:

ADC of the following formula and its salt, solvate and solvate salt

wherein KSP-L- is a compound of the following formula (II), (IIa), (IIb), (IIc), (IId), (IIe), (IIf) or the following formula (IIg), the binding entity is an antibody and n is a value from 1 to 10:

Formula (IIg):

in

_X1 represents N, _X2 represents N and _X3 represents C;

_X1 represents CH, _X2 represents C and _X3 represents N;

_X1 represents NH, _X2 represents C and _X3 represents C; or

_X1 represents CH, _X2 represents N and _X3 represents C;

A represents CO (carbonyl);

R ¹ represents –L-#1, H, –COOH, –CONHNH ₂ , –(CH ₂ ) _1-3 NH ₂ , –CONZ′′(CH ₂ ) _1-3 NH ₂ and –CONZ′′CH ₂ COOH, wherein Z′′ represents H or NH ₂ ;

R ² and R ⁴ represent H or –L-#1, or R ² and R ⁴ together (forming a pyrrolidine ring) represent -CH ₂ -CHR ¹⁰ - or –CHR ¹⁰ -CH ₂ -, wherein R ¹⁰ represents H or –L-#1;

R ³ represents –L-#1 or C _1-10 -alkyl-, which may be optionally substituted by –OH, O-alkyl, SH, S-alkyl, O-CO-alkyl, O-CO-NH-alkyl, NH-CO-alkyl, NH-CO-NH-alkyl, S(O) _n -alkyl, SO ₂ -NH-alkyl, NH-alkyl, N(alkyl) ₂ or NH ₂ (wherein alkyl is preferably C _1-3 -alkyl);

R ⁵ represents –L-#1, H or F;

R ⁶ and R ⁷ independently of one another represent H, (optionally fluorinated) C _1-3 -alkyl, (optionally fluorinated) C _2-4 -alkenyl, (optionally fluorinated) C _2-4 -alkynyl, hydroxy or halogen;

R ⁸ represents a branched C _1-5 -alkyl group; and

^R9 represents H or F,

wherein one of the substituents R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ¹⁰ represents -L-#1, and

–L- represents the linker and the bond between #1 and the linked antibody,

Where –L- is represented by the following formula

§-(CO)m-L1-L2-§§

in

m is 0 or 1;

§ represents the key to KSP and

§§ represents the bond to the antibody, and

L2 Representative

in

# ¹ refers to the connection point with the sulfur atom of the antibody,

# ² represents the connection point with the group L ¹ ,

And L1 is expressed by the following formula

# ¹ –(NR ¹⁰ ) _n -(G1) _o -G2-# ²

in

R ¹⁰ represents H, NH ₂ or C ₁ -C ₃ -alkyl;

G1 stands for –NHCO- or;

n is 0 or 1;

o is 0 or 1; and

G2 represents a linear or branched hydrocarbon chain having 1 to 100 carbon atoms, which is derived from arylene and/or linear and/or branched and/or cyclic alkylene and may be interrupted one or more times by one or more of the radicals -O-, -S-, -SO-, SO ₂ , -NH-, -CO-, -NHCO-, -CONH-, -NMe-, -NHNH-, -SO ₂ NHNH-, -CONHNH- and a 3- to 10-membered aromatic or non-aromatic heterocycle (preferably) having up to 4 heteroatoms selected from N, O and S or -SO-, where side chains, if present, may be substituted by -NHCONH ₂ , -COOH, -OH, -NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid,

# ¹ is the bond to the KSP inhibitor and # ² is the bond to the conjugation group (eg, L2) of the antibody.

Implementation Plan C:

ADC of the following formula and its salt, solvate and solvate salt

wherein KSP-L- is a compound having the substructure I(sub) shown below, the binder is an anti-TWEAKR antibody (particularly preferably an anti-TWEAKR antibody that specifically binds to amino acid D (D47) at position 47 of TWEAKR (SEQ ID NO: 169), in particular the anti-TWEAKR antibody TPP-2090), an anti-HER2 antibody or an anti-EGRF antibody (preferably nimotuzumab), and n is a value from 1 to 10:

in

#a represents the bond to the rest of the molecule;

R ^1a represents -L-#1, H or -(CH ₂ ) _0-3 Z, wherein Z represents -H, halogen, -NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently of one another represent H, NH ₂ or -(CH ₂ CH ₂ O) _0-3 -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ , COOH or -(CO-NH-CHY ⁴ ) _1-3 COOH, wherein Y ⁴ independently of one another represent linear or branched C _1-6 -alkyl optionally substituted by -NHCONH ₂ , or represent aryl or benzyl optionally substituted by -NH ₂ ;

R ^2a and R ^4a independently represent H, -L-#1, -CO-CHY ⁴ -NHY ⁵ or -(CH ₂ ) _0-3 Z, or R ^2a and R ^4a together (forming a pyrrolidine ring) represent -CH ₂ -CHR ¹⁰ - or -CHR ¹⁰ -CH ₂ -, wherein R ¹⁰ represents -L-#1, H, NH ₂ , COOH, SO ₃ H, SH or OH, and wherein Z represents -H, -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH,

wherein Y ⁴ independently of one another represents linear or branched C _1-6 alkyl optionally substituted by -NHCONH ₂ , or represents aryl or benzyl optionally substituted by -NH ₂ , and Y ⁵ represents H or -CO-CHY ⁶ -NH ₂ , wherein Y ⁶ represents linear or branched C _1-6 -alkyl;

wherein one of the substituents R ^1a , R ^2a , R ^4a or R ¹⁰ represents -L-#1,

–L- represents the linker and #1 represents the bond to the antibody,

Where –L- is represented by the following formula

§-(CO)m-L1-L2-§§

in

m is 0 or 1;

§ represents the key to KSP and

§§ represents the bond to the antibody, and

L2 Representative

in

# ¹ refers to the connection point with the sulfur atom of the antibody,

# ² represents the connection point with the group L ¹ ,

And L1 is expressed by the following formula

# ¹ –(NR ¹⁰ ) _n -(G1) _o -G2-# ²

in

R ¹⁰ represents H, NH ₂ or C ₁ -C ₃ -alkyl;

G1 stands for –NHCO- or;

n is 0 or 1;

o is 0 or 1; and

G2 represents a linear or branched hydrocarbon chain having 1 to 100 carbon atoms, which is derived from arylene and/or linear and/or branched and/or cyclic alkylene and may be interrupted one or more times by one or more of the radicals -O-, -S-, -SO-, SO ₂ , -NH-, -CO-, -NHCO-, -CONH-, -NMe-, -NHNH-, -SO ₂ NHNH-, -CONHNH- and a 3- to 10-membered aromatic or non-aromatic heterocycle (preferably) having up to 4 heteroatoms selected from N, O and S or -SO-, where side chains, if present, may be substituted by -NHCONH ₂ , -COOH, -OH, -NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid,

# ¹ is the bond to the KSP inhibitor and # ² is the bond to the conjugation group (eg, L2) of the antibody.

Implementation Plan D:

ADC of the following formula and its salt, solvate and solvate salt

wherein KSP-L- is a compound of the following formula (II), (IIa), (IIb), (IIc), (IId), (IIe), (IIf), (IIg) or the following formula (IIh), the binding entity is an antibody and n is a value from 1 to 10:

Formula (IIh):

in

_X1 represents N, _X2 represents N and _X3 represents C;

_X1 represents CH, _X2 represents C and _X3 represents N;

_X1 represents NH, _X2 represents C and _X3 represents C; or

_X1 represents CH, _X2 represents N and _X3 represents C;

A represents CO (carbonyl);

R ¹ stands for –L-#1;

R ² and R ⁴ represent H, or R ² and R ⁴ together (forming a pyrrolidine ring) represent -CH ₂ -CHR ¹⁰ - or -CHR ¹⁰ -CH ₂ -, wherein R ¹⁰ represents H;

R ³ represents C 1-10 -alkyl- which may be optionally substituted by –OH, O-alkyl, SH, S-alkyl, O-CO-alkyl, O-CO-NH-alkyl, NH-CO-alkyl, NH-CO-NH-alkyl, S(O) _n -alkyl, SO ₂ -NH-alkyl, NH-alkyl, N(alkyl) ₂ or _{NH 2} (wherein alkyl is preferably C _1-3 -alkyl), or –MOD;

Where –MOD stands for –(NR ¹⁰ ) _n -(G1) _o -G2-H, where

R ¹⁰ represents H or C ₁ -C ₃ -alkyl;

G1 represents –NHCO-, -CONH- or (wherein, if G1 represents –NHCO- or , R ¹⁰ does not represent NH ₂ );

n is 0 or 1;

o is 0 or 1; and

G2 represents a straight-chain and/or branched hydrocarbon radical having 1 to 10 carbon atoms and may be interrupted one or more times by one or more of the groups -O-, -S-, -SO-, SO ₂ , -NR ^y -, -NR ^y CO-, CONR ^y -, -NR ^y NR ^y -, -SO ₂ NR ^y NR ^y -, -CONR ^y NR ^y - (wherein R ^y represents H, phenyl, C ₁ -C ₁₀ -alkyl, C ₂ -C ₁₀ -alkenyl or C ₂ -C ₁₀ -alkynyl, each of which may be substituted by -NHCONH ₂ , -COOH, -OH, -NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid), -CO-, -CR ^x =NO- (wherein R x represents H, C ₁ -C ₃ -alkyl or phenyl), wherein the hydrocarbon chain, including side chains, if present, may be interrupted one or more times by -NHCONH ₂ , -COOH, -OH, -NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid substitution, wherein the group -MOD preferably has at least one group -COOH;

R ⁵ represents H or F;

R ⁶ and R ⁷ independently of one another represent H, (optionally fluorinated) C _1-3 -alkyl, (optionally fluorinated) C _2-4 -alkenyl, (optionally fluorinated) C _2-4 -alkynyl, hydroxy or halogen;

R ⁸ represents a branched C _1-5 -alkyl group; and

^R9 represents H or F,

Where –L- represents the linker and #1 represents the bond to the antibody,

Where –L- is represented by the following formula

§-(CO)m-L1-L2-§§

in

m is 0 or 1;

§ represents the bond with KSP and

§§ represents a bond to the antibody, and

L2 Representative

in

# ¹ refers to the connection point to the sulfur atom of the antibody,

# ² represents the connection point to the group ^L1 ,

And L1 is expressed by the following formula

# ¹ –(NR ¹⁰ ) _n -(G1) _o -G2-# ²

in

R ₁₀ represents H, NH ₂ or C ₁ -C ₃ -alkyl;

G1 stands for –NHCO- or;

n is 0 or 1;

o is 0 or 1; and

G2 represents a linear or branched hydrocarbon chain having 1 to 100 carbon atoms, which is derived from arylene and/or linear and/or branched and/or cyclic alkylene and may be interrupted one or more times by one or more of the groups -O-, -S-, -SO-, SO ₂ , -NH-, -CO-, -NHCO-, -CONH-, -NMe-, -NHNH-, -SO ₂ NHNH-, -CONHNH-, -CR ^x =NO- (wherein R ^x represents H, C ₁ -C ₃ -alkyl or phenyl) and a 3- to 10-membered aromatic or non-aromatic heterocycle (preferably) having up to 4 heteroatoms selected from N, O and S, -SO- or -SO ₂ -, wherein the hydrocarbon chain, including side chains, if present, may be substituted by -NHCONH ₂ , -COOH, -OH, -NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid,

# ¹ is the bond to the KSP inhibitor and # ² is the bond to the conjugation group (eg, L2) of the antibody.

Implementation Plan E:

ADC of the following formula and its salt, solvate and solvate salt

wherein KSP-L- is a compound having the substructure I (sub) shown below, the conjugate is Nimutuzumab and n is a value from 1 to 10:

in

#a represents the bond to the rest of the molecule;

R ^1a represents -L-#1, H or -(CH ₂ ) _0-3 Z, wherein Z represents -H, halogen, -NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently of one another represent H, NH ₂ or -(CH ₂ CH ₂ O) _0-3 -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ , COOH or -(CO-NH-CHY ⁴ ) _1-3 COOH, wherein Y ⁴ independently of one another represent linear or branched C _1-6 -alkyl optionally substituted by -NHCONH ₂ , or represent aryl or benzyl optionally substituted by -NH ₂ ;

R ^2a and R ^4a independently represent H, -L-#1, -CO-CHY ⁴ -NHY ⁵ or -(CH ₂ ) _0-3 Z, or R ^2a and R ^4a together (forming a pyrrolidine ring) represent -CH ₂ -CHR ¹⁰ - or -CHR ¹⁰ -CH ₂ -, wherein R ¹⁰ represents -L-#1, H, NH ₂ , COOH, SO ₃ H, SH or OH, and wherein Z represents -H, -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH,

wherein Y ⁴ independently of one another represents linear or branched C _1-6 alkyl optionally substituted by -NHCONH ₂ , or represents aryl or benzyl optionally substituted by -NH ₂ , and Y ⁵ represents H or -CO-CHY ⁶ -NH ₂ , wherein Y ⁶ represents linear or branched C _1-6 -alkyl;

wherein one of the substituents R ^1a , R ^2a , R ^4a or R ¹⁰ represents -L-#1,

–L- represents the linker and #1 represents the bond to the antibody,

Where –L- is represented by the following formula

§-(CO)m-L1-L2-§§

in

m is 0 or 1;

§ represents the key to KSP and

§§ represents the bond to the antibody, and

L2 Representative

in

# ¹ refers to the connection point with the sulfur atom of the antibody,

# ² refers to the connection point with the group L ¹ ,

And L1 is expressed by the following formula

# ¹ –(NR ¹⁰ ) _n -(G1) _o -G2-# ²

in

R ¹⁰ represents H, NH ₂ or C ₁ -C ₃ -alkyl;

G1 stands for –NHCO- or;

n is 0 or 1;

o is 0 or 1; and

G2 represents a linear or branched hydrocarbon chain having 1 to 100 carbon atoms, which is derived from arylene and/or linear and/or branched and/or cyclic alkylene and may be interrupted one or more times by one or more of the radicals -O-, -S-, -SO-, SO ₂ , -NH-, -CO-, -NHCO-, -CONH-, -NMe-, -NHNH-, -SO ₂ NHNH-, -CONHNH- and a 3- to 10-membered aromatic or non-aromatic heterocycle (preferably) having up to 4 heteroatoms selected from N, O and S or -SO-, where side chains, if present, may be substituted by -NHCONH ₂ , -COOH, -OH, -NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid,

# ¹ is the bond to the KSP inhibitor and # ² is the bond to the conjugation group (eg, L2) of the antibody.

In this embodiment, KSP-L- is particularly preferably a salt of the following formula (IIi) and salts, solvates and solvates thereof:

Formula (IIi):

in

_X1 represents N, _X2 represents N and _X3 represents C; or

_X1 represents N, _X2 represents C and _X3 represents N; or

_X1 represents CH or CF, _X2 represents C and _X3 represents N; or

_X1 represents NH, _X2 represents C and _X3 represents C; or

_X1 represents CH, _X2 represents N and _X3 represents C

(wherein X ₁ represents CH, X ₂ represents C and X ₃ represents N is preferred);

R ¹ represents H, -MOD or -(CH ₂ ) _0-3 Z, wherein Z represents -H, -NHY ³ , -OY ³ , -SY ³ , halogen, -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ , -(CH ₂ CH ₂ O) _0-3 -(CH ₂ ) _0-3 Z' (e.g., -(CH ₂ ) _0-3 Z') or -CH(CH ₂ W)Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, NH ₂ , SO ₃ H, COOH, -NH-CO-CH ₂ -CH ₂ -CH(NH ₂ )COOH or -(CO-NH-CHY ⁴ ) _1-3 COOH, wherein W represents H or OH,

wherein Y ⁴ independently of one another represent linear or branched C _1-6 -alkyl optionally substituted by -NHCONH ₂ , or represent aryl or benzyl optionally substituted by -NH ₂ ;

R ² represents H, -MOD, -CO-CHY ⁴ -NHY ⁵ or -(CH ₂ ) _0-3 Z, or R ² and R ⁴ together (forming a pyrrolidine ring) represent –CH ₂ -CHR ¹⁰ - or -CHR ¹⁰ -CH ₂ -, wherein R ¹⁰ represents H, NH ₂ , SO ₃ H, COOH, SH or OH;

wherein Z represents -H, halogen, -OY ³ , -SY ³ , NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH;

wherein Y ⁴ independently of one another represents linear or branched C _1-6 alkyl optionally substituted by -NHCONH ₂ , or represents aryl or benzyl optionally substituted by -NH ₂ , and Y ⁵ represents H or -CO-CHY ⁶ -NH ₂ , wherein Y ⁶ represents linear or branched C _1-6 -alkyl;

R ⁴ represents H, -CO-CHY ⁴ -NHY ⁵ or -(CH ₂ ) _0-3 Z, preferably H,

wherein Z represents -H, halogen, -OY ³ , -SY ³ , NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH;

wherein Y ⁴ independently of one another represents linear or branched C _1-6 alkyl optionally substituted by -NHCONH ₂ , or represents aryl or benzyl optionally substituted by -NH ₂ , and Y ⁵ represents H or -CO-CHY ⁶ -NH ₂ , wherein Y ⁶ represents linear or branched C _1-6 -alkyl;

or R ² and R ⁴ together (forming a pyrrolidine ring) represent –CH ₂ -CHR ¹⁰ - or –CHR ¹⁰ -CH ₂ -, wherein R ¹⁰ represents H, NH ₂ , SO ₃ H, COOH, SH or OH;

A represents CO, SO, SO ₂ , SO ₂ NH or CNNH;

R ³ represents –L-#1,

R ⁵ represents H, NH ₂ , NO ₂ , halogen (especially F, Cl, Br), -CN, CF ₃ , -OCF ₃ , -CH ₂ F, -CH ₂ F, SH or -(CH ₂ ) _0-3 Z, wherein Z represents -H, -OY ³ , -SY ³ , halogen, NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH;

^R6 and ^R7 independently of one another represent H, cyano, (optionally fluorinated) _C1-10 -alkyl, (optionally fluorinated) _C2-10 -alkenyl, (optionally fluorinated) _C2-10 -alkynyl, hydroxy, _NO2 , _NH2 , COOH or halogen (especially F, Cl, Br),

R ⁸ represents (optionally fluorinated) C _1-10 -alkyl, (optionally fluorinated) C _2-10 -alkenyl, (optionally fluorinated) C _2-10 -alkynyl, (optionally fluorinated) C _4-10 -cycloalkyl or –(CH ₂ ) _0-2 -(HZ ² ), wherein HZ ² represents a 4- to 7-membered heterocycle having up to 2 heteroatoms selected from N, O and S, wherein each of these groups may be substituted by –OH, CO ₂ H or NH ₂ ;

L represents a linker and #1 represents a bond to the conjugate or a derivative thereof,

Where –MOD stands for –(NR ¹⁰ ) _n -(G1) _o -G2-H, where

R ¹⁰ represents H or C ₁ -C ₃ -alkyl;

G1 represents –NHCO-, -CONH- or (wherein, if G1 represents –NHCO- or , R ¹⁰ does not represent NH ₂ );

n is 0 or 1;

o is 0 or 1; and

G2 represents a straight-chain and/or branched hydrocarbon radical having 1 to 10 carbon atoms and may be interrupted one or more times by one or more of the groups -O-, -S-, -SO-, SO ₂ , -NR ^y -, -NR ^y CO-, CONR ^y -, -NR ^y NR ^y -, -SO ₂ NR ^y NR ^y -, -CONR ^y NR ^y - (wherein R ^y represents H, phenyl, C ₁ -C ₁₀ -alkyl, C ₂ -C ₁₀ -alkenyl or C ₂ -C ₁₀ -alkynyl, each of which may be substituted by -NHCONH ₂ , -COOH, -OH, -NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid), -CO-, -CR ^x =NO- (wherein R x represents H, C ₁ -C ₃ -alkyl or phenyl), wherein the hydrocarbon chain, including side chains, if present, may be interrupted one or more times by -NHCONH ₂ , —COOH, —OH, —NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid, wherein the group —MOD preferably has at least one group —COOH.

Implementation Plan F:

Compounds of the following general formula and their salts, solvates and salts of solvates:

in

_X1 represents N, _X2 represents N and _X3 represents C, or _X1 represents CH, _X2 represents C and _X3 represents N;

R ¹ represents H or -(CH ₂ ) _0-3 Z, wherein Z represents -H, -NHY ³ , -OY ³ , -SY ³ , halogen, -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ , -(CH ₂ CH ₂ O) _0-3 -(CH ₂ ) _0-3 Z' or -CH(CH ₂ W)Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, NH ₂ , SO ₃ H, COOH, -NH-CO-CH ₂ -CH ₂ -CH(NH ₂ )COOH or -(CO-NH-CHY ⁴ ) _1-3 COOH; wherein W represents H or OH;

wherein Y ⁴ independently of one another represent linear or branched C _1-6 -alkyl optionally substituted by -NHCONH ₂ , or represent aryl or benzyl optionally substituted by -NH ₂ ;

R ² and R ⁴ independently represent H, -CO-CHY ⁴ -NHY ⁵ or -(CH ₂ ) _0-3 Z, or R ² and R ⁴ together (forming a pyrrolidine ring) represent -CH ₂ -CHR ¹⁰ - or -CHR ¹⁰ -CH ₂ -, wherein R ¹⁰ represents H, NH ₂ , SO ₃ H, COOH, SH or OH,

wherein Z represents -H, halogen, -OY ³ , -SY ³ , NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH;

wherein Y ⁴ independently of one another represents linear or branched C _1-6 alkyl optionally substituted by -NHCONH ₂ , or represents aryl or benzyl optionally substituted by -NH ₂ , and Y ⁵ represents H or -CO-CHY ⁶ -NH ₂ , wherein Y ⁶ represents linear or branched C _1-6 -alkyl;

A represents CO, SO, SO ₂ , SO ₂ NH or CNNH;

R ³ represents an optionally substituted alkyl, aryl, heteroaryl, heteroalkyl, heterocycloalkyl, preferably C _1-10 -alkyl, C 6-10 -aryl or C _6-10 -aralkyl, C _5-10 -heteroalkyl, C _1-10 -alkyl-OC _6-10 -aryl or C _5-10 -heterocycloalkyl, which may be substituted by 1-3 –OH groups, _1-3 halogen atoms, 1-3 haloalkyl groups (each having 1-3 halogen atoms), 1-3 O-alkyl groups, 1-3 –SH groups, 1-3 –S-alkyl groups, 1-3 –O-CO-alkyl groups, 1-3 –O-CO-NH-alkyl groups, 1-3 –NH-CO-alkyl groups, 1-3 –NH-CO-NH-alkyl groups, 1-3 –S(O) _n -alkyl groups, 1-3 –SO ₂ -NH-alkyl groups, 1-3 –NH-alkyl groups, 1-3 –N(alkyl) groups ₂ groups, 1 to 3 -NH ₂ groups or 1 to 3 -(CH ₂ ) _0-3 Z groups, where Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ , where Y ¹ and Y ² independently of one another represent H, NH ₂ or -(CH ₂ ) _0-3 Z ', and Y ³ represents H, -(CH ₂ ) _0-3 -CH(NHCOCH ₃ )Z ', -(CH ₂ ) _0-3 -CH(NH ₂ )Z ' or -(CH ₂ ) _0-3 Z ', where Z ' represents H, SO ₃ H, NH ₂ or COOH (wherein "alkyl" preferably represents C _1-10 -alkyl);

R ⁵ represents H, F, NH ₂ , NO ₂ , halogen, SH or -(CH ₂ ) _0-3 Z, wherein Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH;

^R6 and ^R7 independently of one another represent H, cyano, (optionally fluorinated) _C1-10 -alkyl, (optionally fluorinated) _C2-10 -alkenyl, (optionally fluorinated) _C2-10 -alkynyl, hydroxy or halogen,

R ⁸ represents (optionally fluorinated) C _1-10 -alkyl, (optionally fluorinated) C _4-10 -cycloalkyl or optionally substituted oxetane; and

R ⁹ represents H, F, CH ₃ , CF ₃ , CH ₂ F or CHF ₂ .

Implementation Plan G:

The present invention also provides a combination/active substance of the following general formula:

Wherein BINDER represents a binder (preferably an antibody) or a derivative thereof (preferably a cysteine residue), preferably an antibody, L represents a linker, WS represents an active substance, preferably a KSP inhibitor, for example a KSP inhibitor according to the invention of one of the formulae (II), (IIa), (IIb), (IIc), (IId), (IIe), (IIf), (IIg), or (IIh), m represents a value from 1 to 2, preferably 1, and n represents a value from 1 to 50, preferably 1.2 to 20, particularly preferably 2 to 8, wherein L has one of the following structures. Here, m is the number of active substance molecules per linker, and n is the average number of active substance/linker conjugates per BINDER. The sum of all WS present in a conjugate molecule is therefore the product of m and n.

WS are active substances that have a local or systemic therapeutic effect in animals, preferably in humans. These active substances generally have a molecular weight of less than 5 kDa, preferably less than 1.5 kDa. Preferred active substances are antiproliferative substances, such as cytotoxic or cytostatic substances. Preferred active substances are cytotoxic substances, angiogenesis inhibitors, cell cycle inhibitors, PI3 kinase or m-TOR inhibitors, inhibitors of the MAPK signaling cascade, HDAC inhibitors, proteasome inhibitors, PARP inhibitors, Wnt/Hedgehog signaling cascade inhibitors, and RNA polymerase inhibitors. Cytotoxic substances are particularly DNA binding or intercalating substances, DNA-alkylating substances, tubulin stabilizing or destabilizing substances, platinum compounds, and topoisomerase I inhibitors. Exemplary DNA binding substances are, for example, anthracyclines, such as doxorubicin or daunorubicin. Exemplary DNA-alkylating substances are, for example, calicheamicin, temozolomide, or cyclophosphamide and derivatives. Exemplary tubulin stabilizing or destabilizing substances are, for example, taxanes, such as paclitaxel, docetaxel, maytansinoide and auristatine, tubulysine, vinca alkaloids, epothilones and derivatives thereof. Examples of maytansinoides are maytansin, maytansinol, DM-1 and DM-4 (see, for example, U.S. Pat. No. 5,208,020). Examples of auristatines are auristatin E, monomethylauristatin E (MMAE), auristatin F, monomethylauristatin F (MMAF) and dolastin (see, in particular, WO 09/117531, WO 2005/081711, WO 04/010957; WO 02/088172 or WO 01/24763). Examples of vinca alkaloids are vincristine and vinblastine. Examples of epothilones are epothilones A, B, C, D, E or F (see in particular WO 98/13375; WO 2004/005269; WO 2008/138561; WO 2009/002993; WO 2009/055562; WO 2009/012958; WO 2009/026177; WO 2009/134279; WO 2010/033733; WO 2010/034724; WO 2011/017249; WO 2011/057805). Examples of platinum compounds are cisplatin and carboplatin. Examples of topoisomerase I inhibitors are camptothecin and derivatives. Examples of angiogenesis inhibitors are MetAP2 inhibitors, such as fumagillol. Examples of cell cycle inhibitors are CDK inhibitors (e.g., BMS-387032 or PD0332991), Rho kinase inhibitors (e.g., GSK429286), PLK inhibitors (e.g., Volasertib), and Aurora kinase inhibitors (e.g., AZD1152 or MLN805Z). Examples of inhibitors of the MAPK signaling cascade are, in particular, MEK inhibitors (e.g., PD0325901), Ras inhibitors, JNK inhibitors, B-Raf inhibitors (e.g., SB590885), or p38 MAPK inhibitors (e.g., SB202190). Examples of HDAC inhibitors are Belinostat and Givinostat. Examples of PARP inhibitors are Iniparib and Olaparib. Examples of RNA polymerase inhibitors are Amanita toxins (e.g., α-Amantin, Amanitin, and Amanullin). Particularly preferred active substances are Vinca alkaloids, Auristatine, Maytansinoide, Tubulysine, Duocarmycine, kinase inhibitors, MEK inhibitors and KSP inhibitors.

Here, L represents one of the following formulas A3 or A4

wherein # ¹ refers to the point of attachment to the sulfur atom of the binding body, # ² refers to the point of attachment to the active substance, x represents 1 or 2, and ^R22 represents COOH, COOR, COR (wherein R represents in each case _C1-3 -alkyl), _CONH2 , Br, preferably COOH.

L1 has the same meaning as above. -L1-#2 is preferably represented by the following formula:

# ³ –(NR ¹⁰ ) _n -(G1) _o -G2-# ²

in

#3 refers to the connection point with the nitrogen atom,

R ¹⁰ represents H, NH ₂ or C ₁ -C ₃ -alkyl;

G1 represents –NHCO-, -CONH- or (wherein, if G1 represents NHCO or, R10 does not represent NH ₂ ),

n is 0 or 1;

o is 0 or 1; and

G2 represents a linear or branched hydrocarbon chain having 1 to 100 carbon atoms, which is derived from arylene and/or linear and/or branched and/or cyclic alkylene and may be substituted by groups -O-, -S-, -SO-, SO ₂ , -NR ^y -, -NR ^y CO-, -C(NH)NR ^y -, CONR ^y -, -NR ^y NR ^y -, -SO ₂ NR ^y NR ^y -, -CONR ^y NR ^y - (wherein R ^y represents H, phenyl, C ₁ -C ₁₀ -alkyl, C ₂ -C ₁₀ -alkenyl or C ₂ -C ₁₀ -alkynyl, each of which may be substituted by NHCONH ₂ , -COOH, -OH, -NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid), -CO-, -CR ^x =NO- (wherein R ^x represents H, C ₁ -C ₃ -alkyl or phenyl) and/or one or more interruptions one or more times by a 3- to 10-membered aromatic or non-aromatic heterocycle (preferably) having up to 4 heteroatoms selected from N, O and S, -SO- or -SO ₂ -, wherein the hydrocarbon chain, including the side chains, if present, may be substituted by NHCONH ₂ , -COOH, -OH, -NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid.

The further interrupting group in G2 is preferably

wherein Rx represents H, C ₁ -C ₃ -alkyl or phenyl.

In the conjugates according to the invention or in the mixtures of the conjugates according to the invention, the bond to the cysteine residue of the binder is particularly preferably present as one of the two structures of the formula A3 or A4 to an extent of preferably more than 80%, particularly preferably more than 90% (in each case based on the total number of bonds of the linker to the binder).

Conjugates containing linkers of formula A3 or A4 can be obtained by coupling the conjugate to the corresponding bromo derivatives of formula A3' and A4':

These bromo derivatives of formula A3' or A4' can be obtained by reacting _{HOOCCH2CHBrCOOR 22} or _{HOOCCHBrCH2COOR 22} with an amine group of a conjugate as exemplified in the following Schemes 30 to 32.

Figure 30:

[a] 2-Bromo-1-ethylpyridinium tetrafluoroborate (BEP), DCM, pyridine, RT; b) zinc chloride, trifluoroethanol, 50°C, EDTA; c) 3-4 equivalents of TCEP, PBS buffer; d) PBS buffer, 20h RT.]

Figure 31:

[a] 2-Bromo-1-ethylpyridinium tetrafluoroborate (BEP), DCM, pyridine, RT; b) zinc chloride, trifluoroethanol, 50°C, EDTA; c) 3-4 equivalents of TCEP, PBS buffer; d) PBS buffer, 20h RT.]

Implementation Plan H:

The present invention also provides a combination/active substance of the following general formula:

Here, BINDER represents a binder (preferably an antibody) or a derivative thereof (preferably a cysteine residue), preferably an antibody; L represents a linker; WS represents an active substance, preferably a KSP inhibitor, for example a KSP inhibitor according to the invention of one of the formulae (II), (IIa), (III), or (IIIa); m represents a value from 1 to 2, preferably 1; and n represents a value from 1 to 50, preferably 1.2 to 20, particularly preferably 2 to 8, wherein L has one of the following structures. Here, m is the number of active substance molecules per linker, and n is the average number of active substance/linker conjugates per BINDER. The sum of all WS present in a conjugate molecule is therefore the product of m and n.

Here, L stands for:

wherein # ¹ refers to the point of attachment to the sulfur atom of the binding partner, # ² refers to the point of attachment to the active substance, and R ²² represents COOH, COOR, COR (wherein R in each case represents C _1-3 -alkyl), CONH ₂ , Br, preferably COOH. The bond to the sulfur atom of the binding partner may thus have one of the following structures:

In the case of active substance/binder conjugates containing more than one active substance molecule WS per active substance/binder conjugate, both structures according to formula A1 and/or A2 may be present in the active substance/binder conjugate. Since the active substance/binder conjugates of the present invention may be a mixture of different active substance/binder conjugates, such mixtures may also contain active substance/binder conjugates of formula A1 or formula A2, and active substance/binder conjugates of formulas A1 and A2.

_L5 is a group selected from -( _CH2 ) _m- ( ^CHRS ) _n- ( _OCH2CH2 ) _o- (X) _p- ( _CH2 ) _q- , wherein m, n, o, p, and q independently have the following values: _m = 0-10; n = 0 or 1; o = 0-10; p = 0 or 1; and q = 0-10, wherein m + n + o = 1-15, preferably 1-6. X represents a 5- or 6-membered aromatic or non-aromatic heterocyclic or carbocyclic ring, preferably _-C6H4- or _{-C6H10- .} ^RS represents an acid group, preferably -COOH or _{SO3H .}

L ₆ is a group selected from —CONH—, —OCONH—, —NHCO—, —NHCOO—, or —NHCO—, wherein r is 1, 2, or 3.

L ₇ is a single bond or a radical selected from a linear or branched hydrocarbon chain having 1 to 100 (preferably 1 to 10) carbon atoms, which is derived from an arylene group and/or a linear and/or branched and/or cyclic alkylene group and which may be substituted by a radical -O-, -S-, -SO-, SO ₂ , -NR ^y -, -NR ^y CO-, -C(NH)NR ^y -, CONR ^y -, -NR ^y NR ^y -, -SO ₂ NR ^y NR ^y -, -CONR ^y NR ^y - (wherein R ^y represents H, phenyl, C ₁ -C ₁₀ -alkyl, C ₂ -C ₁₀ -alkenyl or C ₂ -C ₁₀ -alkynyl, each of which may be substituted by NHCONH ₂ , -COOH, -OH, -NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid), -CO-, -CR ^x = NO- (wherein R ^x represents H, C ₁ -C ₃ -alkyl or phenyl) and/or one or more interrupted one or more times by a 3- to 10-membered, preferably 5- to 10-membered, aromatic or non-aromatic heterocycle (preferably) having up to 4 heteroatoms selected from N, O and S, -SO- or -SO ₂ -, wherein the hydrocarbon chain, including the side chains, if present, may be substituted by -NHCONH ₂ , -COOH, -OH, -NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid.

L ₅ is preferably a group -(CH ₂ ) _m -(CHR ^S ) _n -(OCH ₂ CH ₂ ) _o -(X) _p -(CH ₂ ) _q -, where m = 1-3, n = 0, o = 0-7, p = 0 and q = 0 or 1. Particularly preferred is a group -(CH ₂ ) _m -(CHR ^S ) _n -(OCH ₂ CH ₂ ) _o -(X) _p -(CH ₂ ) _q -, where m = 1 or 2, n = 0, o = 0 or 1, p = 0 and q = 0 or 1.

L ₆ is preferably a group selected from —CONH— and —NHCO—.

L ₇ is preferably a single bond or -[(CH ₂ ) _x -(X ⁴ ) _y ] w -(CH ₂ ) _z -,

in

w = 0 to 20;

x = 0 to 5;

y = 0 or 1;

z = 1 to 5; and

X ⁴ represents –O-, -CONH-, –NHCO- or .

L ₇ is particularly preferably a single bond or a group —[(CH ₂ ) _x —NHCO—]], where x=1-5.

_-L5 - _L6 - _L7- particularly preferably represents -( _CH2 ) _m- ( ^CHRS ) _n- (OCH2CH2) _{o- ( X} ) _p- ( _CH2 ) _q -NHCO-[( _CH2 ) _x -NHCO-]], where m=1 or 2, n=0, o=0 or 1, p=0, and q=0 or 1, and x=1-5.

However, these two structures may also coexist in the conjugate of the present invention.

According to the present invention, these conjugates/active substance conjugates can be prepared from compounds of the formula

wherein L has the following formula A':

A' is preferably converted to A by stirring in a pH buffer having a pH of 7.5 to 8.5, preferably 8, at a temperature below 37°C, preferably 10 to 25°C, for up to 40 hours, preferably 1 to 15 hours.

Implementation Plan 1:

The antibody conjugate of the following formula

in

R2, R4 and R5 represent H;

R3 represents –CH ₂ OH;

R1 stands for –L1-L2-BINDER, where

L1 Representative

Where #2 represents the connection point with L2, and #1 represents the connection point with another connection;

and L2 represents one or both of the structures of the following formulae A5 and A6:

in

# ¹ refers to the connection point with the sulfur atom of the bond.

# ² refers to the point of connection with the group L ¹ , and

R ²² represents COOH, COOR, COR, CONHR (wherein R in each case represents C 1-3 -alkyl), CONH ₂ , preferably COOH.

In the conjugates according to the invention or in the mixtures of the conjugates according to the invention, bonds to the cysteine residues of the binder are present to an extent of preferably more than 80%, particularly preferably more than 90% (in each case based on the total number of bonds of the linker to the binder), particularly preferably one of the two structures of the formula A5 or A6.

Here, the structures of the formula A5 or A6 are usually present together, preferably in a ratio of 60:40 to 40:60, based on the number of bonds to the combined body. The remaining bonds are then present as the following structure

The binding agent is preferably a binding protein or peptide, particularly preferably a human, humanized, or chimeric monoclonal antibody or antigen-binding fragment thereof, in particular an anti-TWEAKR antibody or antigen-binding fragment thereof or an anti-EGFR antibody or antigen-binding fragment thereof. Particularly preferred are anti-TWEAKR antibodies that specifically bind to amino acid D (D47) at position 47 of TWEAKR (SEQ ID NO: 169), in particular the anti-TWEAKR antibody TPP-2090 or the anti-EGFR antibodies cetuximab or nimotuzumab. Alternatively, a cysteine residue may be present in the binding agent.

Therapeutic uses

Hyperproliferative diseases for which the compounds of the invention can be used include, in particular, cancer and tumor diseases. In the present invention, these are understood to mean, in particular, but not limited to, the following diseases: breast cancer and breast tumors (including ductal and lobular forms, also in situ ), respiratory tract tumors (small cell and non-small cell carcinomas, bronchogenic carcinomas), brain tumors (e.g. brain stem tumors and hypothalamic tumors, astrocytomas, ependymomas, glioblastomas, gliomas, medulloblastomas, meningiomas and neuroectodermal and pineal tumors), digestive organ tumors (esophageal, stomach, gallbladder, small intestine, large intestine, rectum and anal cancer), liver tumors (especially hepatocellular carcinoma, cholangiocarcinoma and mixed hepatocellular and cholangiocarcinoma), tumors of the head and neck region (carcinomas of the larynx, hypopharynx, nasopharynx, oropharynx, lips and oral cavity, oral melanoma), skin tumors (basal cell carcinoma, spinaliome, squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, non-melanoma skin carcinoma, Merkel cell skin cancer, mast cell tumor), tumors of supporting connective tissue (especially soft tissue sarcoma, osteosarcoma, malignant fibrous histiocytoma, chondrosarcoma, fibrosarcoma, angiosarcoma, leiomyosarcoma, liposarcoma, lymphosarcoma, and rhabdomyosarcoma), eye tumors (especially intraocular melanoma and retinoblastoma), tumors of the endocrine and exocrine glands (e.g., thyroid and parathyroid, pancreatic and salivary gland cancers, adenocarcinomas), urinary tract tumors (tumors of the bladder, penis, kidney, renal pelvis, and ureter), and reproductive organ tumors (endometrial, cervical, ovarian, vaginal, vulvar, and uterine cancer in women and prostate and testicular cancer in men). These also include proliferative blood diseases both in solid form and as circulating cells of the blood, lymphatic system and spinal cord, such as leukemias, lymphomas and myeloproliferative disorders, for example acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia and hairy cell leukemia and AIDS-related lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, Burkitt's lymphoma and lymphomas of the central nervous system.

These diseases, which are well characterized in humans, also occur with comparable etiology in other mammals and are likewise treatable there with the compounds of the invention.

The treatment of the above-mentioned cancerous diseases with the compounds of the present invention includes the treatment of solid tumors as well as the treatment of metastatic or circulating forms thereof.

In the present invention, the term "treat" is used in the conventional sense and refers to the care, nursing and supervision of a patient in order to combat, alleviate, lessen or relieve a disease or health disorder and improve living conditions impaired by the disease, such as in the case of cancer.

The present invention therefore also provides the use of the compounds according to the invention for the treatment and/or prophylaxis of diseases, in particular the diseases mentioned above.

The present invention also provides the use of the compounds of the present invention for the manufacture of a medicament for treating and/or preventing diseases, in particular the diseases mentioned above.

The present invention further provides the use of the compounds according to the invention in a method for the treatment and/or prophylaxis of diseases, in particular the diseases mentioned above.

The present invention further provides a method for the treatment and/or prevention of diseases, in particular the diseases mentioned above, using an effective amount of at least one compound according to the invention.

The compounds of the invention can be used alone or, if desired, in combination with one or more other pharmacologically active substances, provided that such a combination does not cause undesirable and unacceptable side effects. The present invention therefore also provides medicaments containing at least one compound of the invention and one or more additional active substances, in particular for the treatment and/or prevention of the above-mentioned diseases.

For example, the compounds of the present invention may be combined with known anti-hyperproliferative, cytostatic or cytotoxic substances for the treatment of cancer. Examples of suitable combination active substances include:

131I-chTNT, Abarelix, Abiraterone, Aclarubicin, Afatinib, Aflibercept, Aldesleukin, Alemtuzumab, Alisertib, Alitretinoin, Alpharadin (Radium-223 Chloride), Hexamethylmelamine, Aminoglutethimide, AMP-514, Amrubicin, Amsacrine, Anastrozole, Arglabin, Arsenic Trioxide, Asparaginase, AT9283, Axitinib, Azacitidine, Basiliximab, Belotecan, Bendamustine, Bevacizumab, Bexarotene, Bicalutamide, Bisantrene, Bleomycin, BMS-936559, Bosutinib, Bortezomib, Brentuximab Vedotin, buserelin, busulfan, cabazitaxel, cabozantinib, leucovorin, leucovorin, capecitabine, carboplatin, carfilzomib (a proteasome inhibitor), carmofur, carmustine, catumaxomab, celecoxib, cimoleukin, cetuximab, chlorambucil, chlormadinone acetate, nitrogen mustard, cisplatin, cladribine, clodronic acid, clofarabine, copanlisib (BAY 80-6946), Crisantaspase, Crizotinib, Cyclophosphamide, CYC116, Cyproterone acetate, Cytarabine, Dacarbazine, Actinomycin, Darbepoetin alfa, Dabrafenib, Darusseritin, Dasatinib, Daunomycin, Decitabine, Degarelix, Denileukin-Diftitox, Denosumab, Deslorelin, Spironium bromide, Docetaxel, Doxifluridine, Doxorubicin, Doxorubicin + Estrone, eculizumab, edrecolomab, elliptonium acetate, idrapopag, endostatin, ENMD-2076, enocitabine, epirubicin, cyclothioandrostol, epoetin alfa, epoetin beta-lactam, epoetin platinum, eribulin, erlotinib, estradiol, estramustine, etoposide, everolimus, exemestane, fadrozole, filgrastim, fludarabine, fluorouracil, flutamide, formestane, fotemustine, fulvestrant, gallium nitrate, ganirelix, gefitinib, gemcitabine, gemtuzumab, Glutoxim, goserelin, histamine dihydrochloride, histrelin, hydroxyurea, iodine I-125 particles, ibandronic acid, ibritumomab tiuxetan, ibrutinib, idarubicin, ifosfamide, imatinib, imiquimod, INCB243 60. Improsulfan, interferon-α, interferon-β, interferon-γ, ipilimumab, irinotecan, ixabepilone, lambrolizumab, lanreotide, lapatinib, lenalidomide, levograstim, lentinan, letrozole, leuprorelin, levamisole, lisuride, lobaplatin, lomustine, lonidamine, masoprocol, medroxyprogesterone acetate, megestrol acetate, melphalan, melastane, mercaptopurine, methotrexate, methoxsalen, methyl aminolevulinate, methyltestosterone, mifatide, miltefosine, miplatin, dibromomannitol, mitoguanidine, dibromodulanol, mitomycin, mitotane, mitoxantrone, MLN-8054, Mps1 inhibitor (disclosed in WO2013/08757) 9, in particular Example 01.01, WO2014/131739, in particular Example 2), nedaplatin, nelarabine, nemorubicin, nilotinib, nilutamide, nimotuzumab, nimustine, diamine nitrazepam, Nivolumab, NMS-P715, NMS-P937, ofatumumab, omeprazole, oprelvekin, oxaliplatin, p53 gene therapy, paclitaxel, Palbociclib, Palifermin, palladium-103 particles, pamidronate, panitumumab, pazopanib, pegaspargase, PEG-beta-epoetin (methoxy-PEG-beta-epoetin), pegfilgrastim, Peg-interferon-α-2b, pemetrexed, pentazocine, Pentostatin, Peplomycin, Perfosfamide, Lysostamicin, Pirarubicin, Plexafor, Plicamycin, Polyglucosamine, Polyestradiol Phosphate, Polysaccharide-K, Ponatinib, Porfimer-Sodium, Pralatrexate, Prednimustine, Procarbazine, Quinagolide, R763, Raloxifene, Raltitrexed, Ranustine, Razoxane, Refametinib, Regorafenib, Risedronate, Rituximab, Romidepsin, Romilastin, Roninciclib, Ruxolitinib, Sargramostim, Sipuleucel-T, Sizosulose, Sobuzoxane, Glycidazole Sodium, SNS-314, Sorafenib, Streptozotocin, Sunitinib, Talaporfin, Tamibarotene, Tamoxifen, Tasonamine, Tesileukin, Tegafur, Tegafur + Gimeracil + Otracillin, temoporfin, temozolomide, temsirolimus, teniposide, testosterone, tetrofosmin, thalidomide, thiotepa, thymalfasin, TKM-PLK1, thioguanine, tocilizumab, topotecan, toremifene, tositumomab, tozasertib, trabectedin, trametinib, trastuzumab, trastuzumab emtansine, sulsulfuron, tretinoin, trilostane, triptorelin, trolofosamide, tryptophan, ubenimex, valrubicin, vandetanib, vapreotide, vemurafenib, vinblastine, vincristine, vindesine, vinflunine, vinorelbine, volasertib, vorinostat, Vorozol, XL228, yttrium-90 glass microspheres, nestatin, nestatin ester, zoledronic acid, zorubicin.

Furthermore, the compounds of the invention can be combined, for example, with binding bodies which can bind, for example, to the following targets: OX-40, CD137/4-1BB, DR3, IDO1/IDO2, LAG-3, CD40.

Furthermore, the compounds of the present invention may also be used in combination with radiation therapy and/or surgery.

In general, the following objectives are pursued with the compounds of the present invention in combination with other cytostatic or cytotoxic agents:

● improved efficacy in slowing down tumor growth, reducing its size or even completely eliminating it compared to treatment with a single active substance;

● the possibility of using chemotherapy drugs at lower doses than in monotherapy;

● Therapy is likely to be better tolerated with fewer side effects than individual medications;

● The possibility of treating a wider spectrum of tumor diseases;

● Achieve higher treatment response rates;

● Longer patient survival compared to today’s standard treatments.

Furthermore, the compounds of the present invention may also be used in combination with radiation therapy and/or surgery.

The invention also provides medicaments which comprise at least one compound according to the invention, generally together with one or more inert, non-toxic, pharmaceutically acceptable adjuvants, and their use for the purposes mentioned above.

The compounds according to the invention can act systemically and/or locally. For this purpose, they can be administered by suitable means, for example parenterally, possibly by inhalation or as an implant or stent.

The compounds according to the invention can be administered in administration forms suitable for these administration routes.

Parenteral administration can bypass the reabsorption step (e.g., intravenous, intraarterial, intracardial, intraspinal, or intralumbar) or include reabsorption (e.g., intramuscular, subcutaneous, intradermal, transdermal, or intraperitoneal). Suitable administration forms for parenteral administration include injections and infusions in the form of solutions, suspensions, emulsions, or lyophilized products. Parenteral administration, particularly intravenous administration, is preferred.

In general, it has been found advantageous in the case of parenteral administration to administer an amount of about 0.001 to 1 mg/kg, preferably about 0.01 to 0.5 mg/kg body weight, to achieve effective results.

However, it may be necessary to deviate from the indicated amounts as appropriate, depending, inter alia, on body weight, route of administration, individual response to the active substance, properties of the formulation and time or interval of administration. Thus, in some cases, less than the above-mentioned minimum amount may be sufficient, while in other cases the upper limit mentioned may have to be exceeded. In the case of administering larger amounts, it may be advisable to divide them into several individual doses throughout the day.

Example

The following examples illustrate the present invention, but the present invention is not limited to these examples.

Unless otherwise indicated, percentages in the following tests and examples are percentages by weight; parts are parts by weight. Solvent ratios, dilution ratios and concentrations of liquid/liquid solutions are in each case based on volume.

Synthesis route:

To illustrate the examples, the following schemes show exemplary synthetic pathways for producing the examples:

Scheme 1: Synthesis of cysteine-linked ADC

Scheme 2: Synthesis of cysteine-linked ADC

Scheme 3: Synthesis of cysteine-linked ADC

Scheme 4: Synthesis of cysteine-linked ADC

Scheme 5: Synthesis of lysine-linked ADC

a) Triphosgene, THF, under argon

Scheme 6: Synthesis of lysine-linked ADC

Diagram 7

[a): EDCI, HOBT, diisopropylethylamine, RT; b) ethanol, piperidine, methylamine, water, RT; c) HATU, diisopropylethylamine, RT; d) TFA, DCM, RT]

Diagram 8

[a): For example, EDCI, HOBT, diisopropylethylamine, DMF, RT; b) For example, DCM/TFA 20:1, RT; c) For example, HATU, diisopropylethylamine, DMF, RT; d) For example, TFA, DCM, RT]

Diagram 9

[a] e.g. 2-bromo-1-ethylpyridinium tetrafluoroborate, diisopropylethylamine, DCM, RT; b) e.g. 2M LiOH solution, THF, water, RT, HPLC separation of regioisomers; c) e.g. EDCI, HOBT, diisopropylethylamine, DMF, RT; d) e.g. TFA, DCM, RT]

Figure 10

[a): For example, H ₂ , Pd-C, EtOH, RT; b) For example, p-nitrobenzyl bromide, K ₂ CO ₃ , DMF; c) For example, ethanol, 40% aqueous methylamine solution, 50°C; d) For example, disodium dithionite, THF, water, 50°C; e) For example, HATU, diisopropylethylamine, DMF, RT; f) For example, piperidine, 40% aqueous methylamine solution, ethanol, 50°C; g) For example, diisopropylethylamine, DMF, RT; h) For example, piperidine, DMF, RT; i) TFA, DCM, RT]

Diagram 11

[a] For example, Et ₃ N, DMF, RT; b) For example, H ₂ , Pd-C, EtOH/ethyl acetate/THF (1:1:1), RT; c) For example, 4-methylmorpholine, DMF, RT; d) For example, HATU, HOAt, diisopropylethylamine, DMF, RT; e) For example, TFA, RT]

Diagram 12

[a] For example, NaBH(OAc) ₃ , HOAc, dichloromethane, RT; b) For example, chloroacetyl chloride, NEt ₃ , DCM, RT; c) For example, Cs ₂ CO ₃ , DMF, 50° C.; d) For example, 1-(2-aminoethyl)-1H-pyrrole-2,5-dione hydrochloride (1:1), T3P ^(R) , diisopropylethylamine, MeCN, RT; e) For example, TFA, RT]

Diagram 13

[a] For example, methanesulfonyl chloride, NEt ₃ , dichloromethane, 0° C.; b) For example, NaN ₃ , DMF, 40° C.; c) For example, H ₂ , Pd-C, EtOH/ethyl acetate (1:1), RT; d) For example, TBAF, THF, RT; e) For example, 1-({[2-(trimethylsilyl)ethoxy]carbonyl}oxy)pyrrolidine-2,5-dione, NEt ₃ , CaCO ₃ , 1,4-dioxane, RT; f) For example, N-chlorosuccinimide, TEMPO, tetra-n-butylammonium chloride, chloroform, 0.05 N potassium carbonate/0.05 N sodium bicarbonate solution (1:1); g) For example, NaBH(OAc) ₃ , HOAc, dichloromethane, RT; h) For example, chloroacetyl chloride, NEt ₃ , DCM, RT; i) For example, TBAF, THF, water, RT; j) For example, 4-methylmorpholine, DMF, RT; k) For example, TFA, RT]

Diagram 14

[a] For example, formaldehyde, _Na2CO3 , water, RT; b) _For example, _Ac2O , pyridine, THF, RT; c) For example, di-tert-butyl malonate, KOtBu, THF, RT; d) For example, _LiBH4 , THF, RT; e) For example, TBDMSCl, imidazole, DCM, RT; f] For example, Dess-Martin periodinane, DCM; g) For example, sodium triacetoxyborohydride, AcOH, DCM, RT; h) For example, _nBu4NF , THF, RT; i) For example, _SOCl2 , THF, RT; j) For example, AcSK, _nBu4NI , DMF, 90°C; k) For example, NaOH, MeOH, THF, RT; l) For example, TCEP, dioxane, RT; m) For example, separation of epimers; n) For example, 6N hydrochloric acid, THF, RT o) For example, Mal-dPEG(3)-Mal, PBS buffer, ACN, RT]

Figure 15

[a] For example, Mal-dPEG(3)-Mal, PBS buffer, ACN, RT]

Diagram 16

[a): For example, BF ₃ OEt ₂ , THF, 0° C.; b): For example, isoamyl nitrite, −10° C., 0.5 h; c]: For example, methyl 2-chloro-3-oxobutanoate, pyridine, water, −5° C.; d): For example, NEt ₃ , toluene, RT; e): For example, Et ₃ SiH, TFA, RT; f): For example, LiBH ₄ , THF, 60° C.; g): For example, Dess-Martin periodinane, DCM, RT; h): For example, (R)-(+)-methyl-2-propanesulfenamide, titanium(IV) isopropoxide, THF, RT; i): For example, tert-BuLi, pentane, THF, −78° C.; j): For example, HCl in dioxane, THF, MeOH, RT; k): For example, 3-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)propanal, NaB(OAc) ₃ H, AcOH, DCM, RT; l): for example, 2-chloro-2-oxoethyl acetate, NEt ₃ , DCM, RT; m): for example, methylamine, water, EtOH, 50° C.]

Diagram 17

[a): For example, tert-butyl N-(tert-butoxycarbonyl)-5-oxo-L-norvaline, NaB(OAc) ₃ H, AcOH, DCM, RT; b): For example, 2-chloro-2-oxoethyl acetate, NEt ₃ , DCM, RT; c): For example, methylamine, water, EtOH, 60° C.; d): For example, THF, DCM, 50° C.; e): For example, Boc ₂ O, NEt ₃ , DCM, RT; f): For example, trifluoroacetic acid/1-(2-aminoethyl)-1H-pyrrole-2,5-dione (1:1), HATU, diisopropylethylamine, DMF, RT; f): For example, TFA, DCM, RT]

Scheme 18: Synthesis of cysteine-linked ADC

Scheme 19: Synthesis of cysteine-linked ADC

Scheme 20: Synthesis of cysteine-linked ADC

Scheme 21: Synthesis of cysteine-linked ADC

Diagram 22

[a] For example, sodium triacetoxyborohydride, acetic acid, DCM, RT; b) For example, acetoxyacetyl chloride, NEt ₃ , DCM, RT; c) For example, LiOH, THF/water, RT; d) For example, H ₂ , Pd-C, EtOH, RT; e) For example, Teoc-OSu, NEt ₃ , dioxane, RT; f) For example, Fmoc-Cl, diisopropylethylamine, dioxane/water 2:1, RT]

Figure 23

[a] For example, sodium triacetoxyborohydride, acetic acid, DCM, RT; b) For example, acetoxyacetyl chloride, NEt ₃ , DCM, RT; c) For example, LiOH, methanol, RT; d) For example, TFA, DCM, RT; e) For example, Boc ₂ O, diisopropylethylamine, DCM, RT]

Figure 24

[a] For example, benzyl bromide, _Cs2CO3 , _DMF , RT; b) For example, Pd(dppf) _{2Cl2 ,} DMF, _Na2CO3 , 85° _C ; c) For example, _LiAlH4 , THF, 0°C; _MnO2 , DCM, RT; d) For example, Ti(iOPr) ₄ , THF, RT; e) For example, tBuLi, THF, -78°C; MeOH, _NH4Cl ; f] For example, HCl/1,4-dioxane]

Scheme 25: Synthesis of cysteine-linked ADC

Scheme 26: Synthesis of Cysteine-linked ADCs via Succinamide Hydrolysis

This method is particularly useful for ADCs where L1 = _CH2 to convert these ADCs into open-chain linked forms.

Figure 27:

[a] Sodium triacetoxyborohydride, acetic acid, DCM, RT; b) Acetoxyacetyl chloride, diisopropylethylamine, DCM, RT; c) LiOH, MeOH, RT; d) Trifluoroacetic acid/1-(2-aminoethyl)-1H-pyrrole-2,5-dione (1:1) HATU, DMF, diisopropylethylamine, RT; e) Zinc chloride, trifluoroethanol, 50°C, EDTA.]

Figure 28:

[a] HATU, DMF, diisopropylethylamine, RT; b) zinc chloride, trifluoroethanol, 50°C, EDTA.]

Figure 29:

[a] Sodium triacetoxyborohydride, acetic acid, DCM, RT; b) Acetoxyacetyl chloride, triethylamine, DCM, RT; c) LiOH, MeOH, RT; d) Trifluoroacetic acid/1-(2-aminoethyl)-1H-pyrrole-2,5-dione (1:1) HATU, DMF, diisopropylethylamine, RT; e) Zinc chloride, trifluoroethanol, 50°C, EDTA.]

Figure 30:

[a] 2-Bromo-1-ethylpyridinium tetrafluoroborate (BEP), DCM, pyridine, RT; b) Zinc chloride, trifluoroethanol, 50°C, EDTA; c) 3-4 equivalents of TCEP, PBS buffer; d) PBS buffer, 20 h RT.]

Figure 31:

[a): 2-bromo-1-ethylpyridinium tetrafluoroborate (BEP), DCM, pyridine, RT; b) zinc chloride, trifluoroethanol, 50°C, EDTA; c) 3-4 equivalents of TCEP, PBS buffer; d) PBS buffer, 20h RT.].

A. Examples

Abbreviations and acronyms:

A431NS human tumor cell line

A549 human tumor cell line

ABCB1 ATP-binding cassette subfamily B member 1 (synonymous with P-gp and MDR1)

abs. pure

Ac acetyl

CAN acetonitrile

aq. containing water, aqueous solution

ATP adenosine triphosphate

BCRP Breast cancer resistance protein, efflux transporter

BEP 2-Bromo-1-ethylpyridinium tetrafluoroborate

Boc tert- Butyloxycarbonyl

br. wide (in NMR)

Bsp. Example

CI Chemical ionization (in MS)

d Doublet (in NMR)

d day

DC thin layer chromatography

DCI Direct Chemical Ionization (in MS)

dd doublet (in NMR)

DMAP 4- N,N -dimethylaminopyridine

DME 1,2-dimethoxyethane

DMEM Dulbecco’s Modified Eagle Medium (standardized nutrient medium for cell culture)

DMF N,N -dimethylformamide

DMSO dimethyl sulfoxide

DPBS, D-PBS, PBS Dulbecco's phosphate buffered saline solution

PBS = DPBS = D-PBS, pH 7.4, from Sigma, No D8537

Composition:

　　　 0.2 g KCl

0.2 g KH ₂ PO ₄ (anhydrous)

8.0 g NaCl

1.15 g Na ₂ HPO ₄ (anhydrous)

Make up to 1 L with H ₂ O

dt doublet triplet (in NMR)

DTT DL-dithiothreitol

d. Th. Theoretical value (in chemical yield)

EDC N' -(3-Dimethylaminopropyl) -N -ethylcarbodiimide hydrochloride

EGFR Epidermal Growth Factor Receptor

EI Electron Impact Ionization (in MS)

ELISA enzyme-linked immunosorbent assay

eq. equivalent

ESI Electrospray ionization (in MS)

ESI-MicroTofq ESI- MicroTofq (mass spectrometer designations Tof = time of flight and q = quadrupole)

FCS fetal calf serum

Fmoc ( 9H -fluoren-9-ylmethoxy)carbonyl

ges. saturation

GTP 5'-guanosine triphosphate

h hour

HATU O -(7-Azabenzotriazol-1-yl)- N,N,N',N' -tetramethyluronium hexafluorophosphate

HCT-116 human tumor cell line

HEPES 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid

HOAc acetic acid

HOAt 1-Hydroxy-7-azabenzotriazole

HOBt 1-Hydroxy- 1H -benzotriazole hydrate

HOSu N -hydroxysuccinimide

HPLC High-pressure liquid chromatography

HT29 human tumor cell line

_IC50 half-maximal inhibitory concentration

i.m. intramuscular, administration into a muscle

i.v. intravenous, giving medicine into a vein

konz. Concentrate

LC-MS liquid chromatography-mass spectrometry

LLC-PK1 cells Lewis lung cancer porcine kidney cell line

L-MDR human MDR1 transfected into LLC-PK1 cells

m multiplet (in NMR)

MDR1 multidrug resistance protein 1

MeCN Acetonitrile

min

MS

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- 2H -tetrazolium bromide

NCI-H292 human tumor cell line

NCI-H520 human tumor cell line

NMM N -methylmorpholine

NMP N -methyl-2-pyrrolidone

NMR nuclear magnetic resonance spectroscopy

NMRI mouse strain, derived from the Naval Medical Research Institute (NMRI)

Nude Mäuse nude mice (test animals)

NSCLC Non-small cell lung cancer

PBS Phosphate-buffered saline

Pd/C activated carbon supported palladium

P-gp P-glycoprotein, transport protein

PNGaseF enzyme for cleaving sugars

quant. Quantitative (yield)

quart (in NMR)

quint quintet (in NMR)

R _f retention index (in DC)

RT Room temperature

_Rt retention time (in HPLC)

s singlet (in NMR)

s.c. subcutaneous, administered under the skin

SCC-4 human tumor cell line

SCC-9 human tumor cell line

SCID Mäuse Severe Combined Immunodeficiency Mice

t triplet (in NMR)

TBAF Tetra-n-butylammonium fluoride

TEMPO (2,2,6,6-tetramethylpiperidin-1-yl)oxy

tert . uncle

TFA trifluoroacetic acid

THF Tetrahydrofuran

T3P ^® 2,4,6-Tripropyl-1,3,5,2,4,6-trioxatriphosphaninane 2,4,6-trioxide

UV spectroscopy

v/v volume ratio (of a solution)

Z benzyloxycarbonyl.

If no temperature is given in the description of a reaction in this disclosure, room temperature should always be assumed.

HPLC and LC-MS methods:

Method 1 (LC-MS ):

Instrument: Waters ACQUITY SQD UPLC system; Column: Waters Acquity UPLC HSS T3 1.8µ 50 x 1 mm; Eluent A: 1 L water + 0.25 mL 99% formic acid; Eluent B: 1 L acetonitrile + 0.25 mL 99% formic acid; Gradient: 0.0 min 90% A → 1.2 min 5% A → 2.0 min 5% A; Oven: 50°C; Flow rate: 0.40 ml/min; UV detection: 208 – 400 nm.

Method 2 (LC-MS):

MS instrument type: Waters Synapt G2S; UPLC instrument type: Waters Acquity I-CLASS; column: Waters, BEH300, 2.1 x 150 mm, C18 1.7 µm; eluent A: 1 L water + 0.01% formic acid; eluent B: 1 L acetonitrile + 0.01% formic acid; gradient: 0.0 min 2% B → 1.5 min 2% B → 8.5 min 95% B → 10.0 min 95% B; oven: 50°C; flow rate: 0.50 ml/min; UV detection: 220 nm.

Method 3 (LC-MS):

MS instrument: Waters (Micromass) QM; HPLC instrument: Agilent 1100 series; column: Agilent ZORBAX Extend-C18 3.0 x 50 mm 3.5 micron; eluent A: 1 liter water + 0.01 mol ammonium carbonate, eluent B: 1 liter acetonitrile; gradient: 0.0 min 98% A → 0.2 min 98% A → 3.0 min 5% A → 4.5 min 5% A; oven: 40°C; flow rate: 1.75 ml/min; UV detection: 210 nm.

Method 4 (LC-MS ):

MS instrument type: Waters Synapt G2S; UPLC instrument type: Waters Acquity I-CLASS; column: Waters, HSST3, 2.1 x 50 mm, C18 1.8 µm; eluent A: 1 L water + 0.01% formic acid; eluent B: 1 L acetonitrile + 0.01% formic acid; gradient: 0.0 min 10% B → 0.3 min 10% B → 1.7 min 95% B → 2.5 min 95% B; oven: 50°C; flow rate: 1.20 ml/min; UV detection: 210 nm.

Method 5 (LC-MS):

Instrument: Waters ACQUITY SQD UPLC system; Column: Waters Acquity UPLC HSS T3 1.8µ 50 x 1 mm; Eluent A: 1 L water + 0.25 mL 99% formic acid; Eluent B: 1 L acetonitrile + 0.25 mL 99% formic acid; Gradient: 0.0 min 95% A → 6.0 min 5% A → 7.5 min 5% A; Oven: 50°C; Flow rate: 0.35 mL/min; UV detection: 210 – 400 nm.

Method 6 (LC-MS ):

Instrument: Micromass Quattro Premier with Waters UPLC Acquity; Column: ThermoHypersil GOLD 1.9 µ 50 x 1 mm; Eluent A: 1 L water + 0.5 mL 50% formic acid; Eluent B: 1 L acetonitrile + 0.5 mL 50% formic acid; Gradient: 0.0 min 97% A → 0.5 min 97% A → 3.2 min 5% A → 4.0 min 5% A; Oven: 50°C; Flow rate: 0.3 ml/min; UV detection: 210 nm.

Method 7 (LC-MS):

Instrument: Agilent MS Quad 6150; HPLC: Agilent 1290; Column: Waters Acquity UPLCHSS T3 1.8 µ 50 x 2.1 mm; Eluent A: 1 L water + 0.25 mL 99% formic acid; Eluent B: 1 L acetonitrile + 0.25 mL 99% formic acid; Gradient: 0.0 min 90% A → 0.3 min 90% A → 1.7 min 5% A → 3.0 min 5% A; Oven: 50°C; Flow rate: 1.20 ml/min; UV detection: 205 – 305 nm.

Method 8 (LC-MS ):

MS instrument type: Waters Synapt G2S; UPLC instrument type: Waters Acquity I-CLASS; column: Waters, HSST3, 2.1 x 50 mm, C18 1.8 µm; eluent A: 1 L water + 0.01% formic acid; eluent B: 1 L acetonitrile + 0.01% formic acid; gradient: 0.0 min 2% B → 2.0 min 2% B → 13.0 min 90% B → 15.0 min 90% B; oven: 50°C; flow rate: 1.20 ml/min; UV detection: 210 nm.

Method 9 : LC-MS-Prep purification method for Examples 181-191 (Method LIND-LC-MS-Prep)

MS instrument: Waters; HPLC instrument: Waters (column Waters X-Bridge C18, 19 mm x 50 mm, 5 µm, eluent A: water + 0.05% ammonia, eluent B: acetonitrile with gradient (ULC); flow rate: 40 ml/min; UV detection: DAD; 210 – 400 nm).

or:

MS instrument: Waters; HPLC instrument: Waters (column Phenomenex Luna 5µ C18(2) 100A, AXIA Tech. 50 x 21.2 mm, eluent A: water + 0.05% formic acid, eluent B: acetonitrile with gradient (ULC); flow rate: 40 ml/min; UV detection: DAD; 210 – 400 nm).

Method 10 : LC-MS analysis method for Examples 181-191 (LIND_SQD_SB_AQ)

MS instrument: Waters SQD; HPLC instrument: Waters UPLC; column: Zorbax SB-Aq (Agilent), 50 mm x 2.1 mm, 1.8 µm; eluent A: water + 0.025% formic acid, eluent B: acetonitrile (ULC) + 0.025% formic acid; gradient: 0.0 min 98%A - 0.9 min 25%A - 1.0 min 5%A - 1.4 min 5%A - 1.41 min98%A - 1.5 min 98%A; oven: 40°C; flow rate: 0.600 ml/min; UV detection: DAD; 210 nm.

Method 11 (HPLC):

Instrument: HP1100 series

Column: Merck Chromolith SpeedROD RP-18e, 50-4.6 mm, catalog number 1.51450.0001, precolumn Chromolith Guard Cartridge Kit, RP-18e, 5-4.6 mm, catalog number 1.51470.0001

Gradient: flow rate 5 ml/min

Injection volume 5 µl

Solvent A: HClO ₄ (70%) in water (4 ml/l)

Solvent B: Acetonitrile

Starting point 20% B

0.50 min 20% B

3.00 min 90% B

3.50 min 90% B

3.51 min 20% B

4.00 min 20% B

Column temperature: 40℃

Wavelength: 210 nm.

All reactants or reagents whose preparation is not explicitly described below were purchased from commonly available sources. For all other reactants or reagents whose preparation is also not described below and which are not commercially available or obtained from commonly unavailable sources, reference is made to published literature describing their preparation.

Raw materials and intermediates:

Intermediate C1

(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropan-1-amine trifluoroacetate (1:1)

The title compound was prepared as described in WO2006/002326.

Intermediate C2

tert-Butyl (2S)-4-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino)-2-[(tert-butoxycarbonyl)amino]butanoate

4.22 g (14.5 mmol) of tert-butyl N-(tert-butoxycarbonyl)-L-homoserine ester was dissolved in 180 ml of dichloromethane, followed by the addition of 3.5 ml of pyridine and 9.2 g (21.7 mmol) of 1,1,1-triacetoxy-1λ ^5,2 -benzidoyl-3(1H)-one. The mixture was stirred at room temperature for 1 hour, then diluted with 500 ml of dichloromethane and extracted twice with 10% sodium thiosulfate solution, followed by two extractions with 5% citric acid and two extractions with 10% sodium bicarbonate solution. The organic phase was separated, dried over magnesium sulfate, and then concentrated under vacuum. The residue was placed in dimethyl ether (DMC), and a mixture of diethyl ether and n-pentane was added. The precipitate was filtered off, and the filtrate was concentrated and lyophilized from acetonitrile/water. This gave 3.7 g (93%) of tert-butyl (2S)-2-[(tert-butoxycarbonyl)amino]-4-oxobutanoate, which was used in the next step without further purification. (R _f value: 0.5 (DCM/methanol 95/5)).

3.5 g (9.85 mmol) of intermediate C1 were dissolved in 160 ml of DCM, and 3.13 g (14.77 mmol) of sodium triacetoxyborohydride and 0.7 ml of acetic acid were added. After stirring at room temperature for 5 minutes, 3.23 g (11.85 mmol) of tert-butyl (2S)-2-[(tert-butoxycarbonyl)amino]-4-oxobutanoate were added, and the preparation was stirred at room temperature for a further 30 minutes. The solvent was then evaporated under vacuum, and the residue was taken up in acetonitrile/water. The precipitated solid was filtered off and dried to yield 5.46 g (84%) of the title compound.

HPLC (Method 11): R _t = 2.5 min;

LC-MS (Method 1): R _t = 1.13 min; MS (ESIpos): m/z = 613 (M+H) ⁺ .

Intermediate C3

(2S)-4-[(Acetoxyacetyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino]-2-[(tert-butoxycarbonyl)amino]butanoic acid

5.46 g (8.24 mmol) of intermediate C2 were dissolved in 160 ml of DCM, followed by the addition of 4.8 ml of triethylamine and 2.2 ml (20.6 mmol) of acetoxyacetyl chloride. The preparation was stirred at room temperature overnight and then concentrated under vacuum. The residue was taken up in ethyl acetate and extracted three times with saturated sodium bicarbonate solution, followed by extraction with saturated sodium chloride solution. The organic phase was dried over sodium sulfate and then concentrated. The residue was purified by column chromatography on a Biotage/Isolera (SNAP 340 g) using an eluent of cyclohexane/ethyl acetate 2:1. This yielded 4.57 g (75%) of the acylated intermediate.

LC-MS (Method 1): R _t = 1.49 min; MS (ESIpos): m/z = 713 (M+H) ⁺ .

1 g (1.36 mmol) of this intermediate was dissolved in 20 ml of DCM, and 20 ml of TFA was added. After stirring at room temperature for 5 hours, the mixture was concentrated and the residue was stirred twice with n-pentane. In each case, the n-pentane was decanted and the remaining solid was dried under high vacuum. This yielded 1.1 g of (2S)-4-[(acetoxyacetyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino]-2-aminobutyric acid/trifluoroacetic acid (1:1). LC-MS (Method 1): R _t = 0.93 min; MS (ESIpos): m/z = 557 (M+H) ⁺ .

0.91 g (1.57 mmol) of this intermediate was dissolved in 70 ml of DCM, and 3.43 g (15.7 mmol) of di-tert-butyl dicarbonate and 4.1 ml of N , N -diisopropylethylamine were added. After stirring at room temperature for 30 minutes, the preparation was diluted with DCM and extracted with 5% citric acid. The organic phase was dried over sodium sulfate and concentrated. The residue was stirred twice with n-pentane and the n-pentane was decanted in each case. The residual solid was lyophilized from acetonitrile/water 1:1 to produce 1.11 g of the title compound.

HPLC (Method 11): R _t = 2.55 min;

LC-MS (Method 1): R _t = 1.3 min; MS (ESIpos): m/z = 657 (M+H) ⁺ .

Intermediate C4

(2S)-2-Amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butanoic acid/trifluoroacetic acid (1:1)

5.46 g (8.24 mmol) of intermediate C2 were dissolved in 160 ml of DCM, and 4.8 ml of triethylamine and 2.2 ml (20.6 mmol) of acetoxyacetyl chloride were added. The preparation was stirred at room temperature overnight and then concentrated under vacuum. The residue was taken up in ethyl acetate and extracted three times with saturated sodium bicarbonate solution and then with saturated sodium chloride solution. The organic phase was dried over sodium sulfate and then concentrated. The residue was purified by column chromatography on Biotage/Isolera (SNAP 340 g) using the eluent cyclohexane/ethyl acetate 2:1. This yielded 4.57 g (75%) of the acylated intermediate.

LC-MS (Method 1): R _t = 1.49 min; MS (ESIpos): m/z = 713 (M+H) ⁺ .

1.5 g (2.035 mmol) of this intermediate were placed in 50 ml of ethanol, and 5.8 ml of a 40% aqueous methylamine solution were added. The preparation was stirred at 50°C for 4 hours and then concentrated. The residue was taken up in DCM and washed twice with water. The organic phase was dried over magnesium sulfate and then concentrated. The residue was dried under high vacuum. This yielded 1.235 mg of this intermediate, which was reacted further without further purification.

1.235 mg (1.5 mmol) of this intermediate was dissolved in 15 ml of DCM and 15 ml of TFA was added. The mixture was stirred at room temperature for 4 hours and then concentrated. The residue was purified by preparative HPLC. The appropriate fractions were concentrated and the residue was lyophilized from acetonitrile. This yielded 1.04 g (quantitative) of the title compound.

HPLC (Method 11): R _t = 1.9 min;

LC-MS (Method 1): R _t = 0.89 min; MS (ESIpos): m/z = 515 (M+H) ⁺ .

Intermediate C5

(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-2-[(tert-butoxycarbonyl)amino]butanoic acid

0.9 g (1.24 mmol) of intermediate C4 was dissolved in 60 ml of DCM, and 2.7 g (12.5 mmol) of di-tert-butyl dicarbonate and 3.3 ml of N , N -diisopropylethylamine were added. After stirring at room temperature for 45 minutes, the preparation was concentrated and the residue was taken up in diethyl ether, and n-pentane was added until it began to become cloudy. The preparation was cooled to 0°C and then decanted. n-Pentane was again added to the residue and decanted. The residual solid was lyophilized from acetonitrile/water 1:1 to yield 0.95 g (quantitative) of the title compound.

HPLC (Method 11): R _t = 2.5 min;

LC-MS (Method 1): R _t = 1.27 min; MS (ESIpos): m/z = 615 (M+H) ⁺ .

Intermediate C6

Trifluoroacetic acid/tert-butyl {(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-1-hydrazino-1-oxobutan-2-yl}carbamate (1:1)

150 mg (0.16 mmol) of intermediate C3 was dissolved in 21 ml of DMF, followed by the addition of 37.2 mg (0.19 mmol) of N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride (EDC), 37 mg (0.243 mmol) of 1-hydroxybenzotriazole, 85 μl of N , N -diisopropylethylamine, and finally 45 mg (0.18 mmol) of commercially available 9H-fluoren-9-ylmethyl hydrazinecarboxylate. The preparation was stirred at room temperature overnight and then concentrated under vacuum. The residue was purified by preparative HPLC. The appropriate fractions were concentrated, and the residue was lyophilized from acetonitrile/water. This yielded 60 mg (41% of theory) of the protected intermediate.

HPLC (Method 11): R _t = 2.9 min;

LC-MS (Method 1): R _t = 1.47 min; MS (ESIpos): m/z = 893 (M+H) ⁺ .

60 mg (0.067 mmol) of this intermediate were dissolved in 19 ml of ethanol, and 681 μl of piperidine and 386 μl of a 40% aqueous methylamine solution were added. The preparation was stirred at 50°C for 18 hours and then concentrated. The residue was taken up in acetonitrile/water in a ratio of 2:1 and adjusted to pH 2 with TFA. The mixture was then reconcentrated and purified by preparative HPLC. The appropriate fractions were concentrated, and the residue was lyophilized from acetonitrile/water. This yielded 25 mg (51% of theory) of the title compound.

HPLC (Method 11): R _t = 2.2 min;

LC-MS (Method 1): R _t = 1.27 min; MS (ESIpos): m/z = 629 (M+H) ⁺ .

Intermediate C7

1-{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-2-[(tert-butoxycarbonyl)amino]butanoyl}hydrazino)acetic acid/trifluoroacetic acid (1:1)

0.2 g (0.305 mmol) of intermediate C3 was dissolved in 80 ml of DCM, and 0.125 g (0.46 mmol) of 2-bromo-1-ethylpyridinium tetrafluoroborate (BEP), 94 mg (0.61 mmol) of commercially available ethyl hydrazinoacetate hydrochloride, and 159 μL of N,N -diisopropylethylamine were added. The mixture was then stirred at room temperature for 1 hour. Ethyl acetate and water were then added to the reaction mixture, and the phases were separated. The organic phase was extracted with saturated sodium chloride solution, dried over magnesium sulfate, filtered, and concentrated. The residue was dried under vacuum and reacted further without purification. To this end, it was taken up in 20 ml of tetrahydrofuran, and 10 ml of water and 3.2 ml of 2N lithium hydroxide solution were added. The preparation was stirred at room temperature for 1 hour and then adjusted to pH 7 using TFA. The preparation was then concentrated, and the residue was purified by preparative HPLC. This allowed the title compound to be separated from its earlier-eluting regioisomer. Combining the corresponding fractions, lyophilizing and drying gave 19.7 g (8% of theory over 2 steps) of the title compound in the form of a colorless foam.

HPLC (Method 11): R _t = 2.4 min;

LC-MS (Method 1): R _t = 1.22 min; MS (ESIpos): m/z = 687 (M+H) ⁺ .

After separation of the regioisomers at the protected intermediate stage, the structural assignment of the regioisomers was performed in a separate experiment by NMR spectroscopy. The protected intermediate of the title compound (ethyl 1-{(2S)-4-[(acetoxyacetyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino]-2-[(tert-butoxycarbonyl)amino]butanoyl}hydrazino)acetate has the following 1H NMR spectrum:

¹ H NMR (500 MHz, DMSO-d ₆ ): d = 7.8 (m, 2H), 7.4-7.2 (m, 6H), 7.08 (m,1H), 6.73 (d, 1H), 5.6 (s, 1H), 5.25 and 4,89 (2d, 2H), 4.89 and 4.77 (2d, 2H),4.62 (t, 1H), 4.32 and 3.78 (2d, 2H), 4.1 (t, 2H), 3.62-3.47 (m), 2.13 (s, 3H),1.41 and 0.72 (2m, 2H), 1.3 (s, 9H), 1.18 (t, 3H), 0.92 (s, 9H).

Intermediate C8

N-{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-2-[(tert-butoxycarbonyl)amino]butanoyl}-β-alanine

293 mg (0.41 mmol) of intermediate C3 were dissolved in 25 ml of DMF, followed by the addition of 144 mg (0.75 mmol) of N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride (EDC), 128 mg (0.83 mmol) of 1-hydroxybenzotriazole, 218 μl of N , N -diisopropylethylamine, and finally 70 mg (0.5 mmol) of commercially available 3-methoxy-3-oxopropan-1-aminium chloride. The preparation was stirred at room temperature for 4 hours and then concentrated under vacuum. The residue was purified by preparative HPLC. The appropriate fractions were concentrated, and the residue was dried under high vacuum. This yielded 177 mg (53% of theory) of the protected intermediate.

HPLC (Method 11): R _t = 2.6 min;

LC-MS (Method 1): R _t = 1.33 min; MS (ESIpos): m/z = 742 (M+H) ⁺ .

177 mg (0.22 mmol) of this intermediate were placed in 20 ml of methanol, and 2.8 ml of 2N lithium hydroxide solution were added. The preparation was stirred at room temperature for 18 hours. It was then concentrated, the residue was placed in water, and the solution was adjusted to pH 5 using 5% citric acid. It was then extracted twice with DCM, and the organic phase was dried over magnesium sulfate and concentrated. The residue was finally lyophilized from acetonitrile/water to yield 133 mg (81% of theory) of the title compound.

HPLC (Method 11): R _t = 2.3 min;

LC-MS (Method 3): R _t = 7.4 min; MS (ESIpos): m/z = 686 (M+H) ⁺ .

Intermediate C9

(6S)-6-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]ethyl}-2,2-dimethyl-4,7-dioxo-3,11,14,17-tetraoxa-5,8-diazaeicosane-20-oic acid

In the first step, 70 mg (0.114 mmol) of intermediate C5 were coupled with 32 mg (0.114 mmol) of tert-butyl 3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy } propanoate in 15 ml of DMF in the presence of 44 mg (0.228 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 35 mg (0.228 mmol) of 1-hydroxy-1H-benzotriazole hydrate and 60 μl of N,N -diisopropylethylamine.

The preparation was stirred at room temperature overnight, and the product was purified by preparative HPLC. 33 mg (33% of theory) of the protected intermediate were isolated. This was stirred with 1.1 ml of trifluoroacetic acid in 11 ml of dichloromethane for 1 hour to yield, after workup, 26 mg (98%) of the fully deprotected compound.

Finally, this intermediate was taken up in 2 ml of DCM and the tert-butoxycarbonyl protecting group was introduced by adding in each case 10 mg of di-tert-butyl dicarbonate and 79 μl of N,N -diisopropylethylamine twice with stirring at room temperature for 3 days. The product was purified by preparative HPLC to yield 16.4 mg (66% of theory) of the title compound.

HPLC (Method 11): R _t = 2.3 min;

LC-MS (Method 1): R _t = 1.22 min; MS (ESIpos): m/z = 818 (M+H) ⁺ .

Intermediate C10

tert-Butyl {3-[{(1R)-1-[1-(3-aminobenzyl)-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]propyl}carbamate

The title compound was prepared from intermediate C1 in six steps: In the first step, 1 g (2.77 mmol) of intermediate C1 and 0.864 g (5 mmol) of tert-butyl (3-oxopropyl)carbamate were combined in 100 ml of methanol, and 400 ml of acetic acid and 1.288 g (13.9 mmol) of borane-pyridine complex were added. The preparation was stirred at room temperature for 3 days. The mixture was then concentrated under vacuum, and the residue was purified by flash chromatography on silica gel (eluent: dichloromethane/ethyl acetate 9:1 -> dichloromethane/methanol 95:5). Concentration of the appropriate fractions and drying under high vacuum yielded 1.255 g (80% of theory) of the N-alkylated intermediate.

LC-MS (Method 1): R _t = 1.0 min; MS (ESIpos): m/z = 513 (M+H) ⁺ .

1.255 g (2.2 mmol) of this intermediate was dissolved in 50 ml of DCM, followed by the addition of 1.2 ml of triethylamine and 0.52 ml (4.85 mmol) of acetoxyacetyl chloride. The mixture was stirred at room temperature overnight and then concentrated under vacuum. The residue was taken up in ethyl acetate and extracted three times with saturated sodium bicarbonate solution and then with saturated sodium chloride solution. The organic phase was dried over sodium sulfate and then concentrated. The residue was purified by preparative HPLC.

This gave 593 mg (41% of theory) of the acylated intermediate.

LC-MS (Method 1): R _t = 1.4 min; MS (ESIpos): m/z = 613 (M+H) ⁺ .

993 mg (0.91 mmol) of this intermediate was dissolved in 100 ml of ethanol and, after adding 60 mg of 10% palladium on activated carbon, hydrogenated at room temperature under standard hydrogen pressure for 3 hours. The catalyst was then filtered off and the solvent removed under vacuum. This yielded 494 mg (91% of theory) of the debenzylated imidazole derivative as a nearly colorless oil. LC-MS (Method 1): R _t = 1.17 min; MS (ESIpos): m/z = 523 (M+H) ⁺ .

Initially, 150 mg (0.25 mmol) of this intermediate was charged to 15 mL of DMF, and 69.2 mg (0.5 mmol) of potassium carbonate was added. After stirring at room temperature for 15 minutes, 60 mg (0.28 mmol) of p-nitrobenzyl bromide was added and stirred overnight. The solvent was then removed under vacuum, and the residue was taken up in ethyl acetate and extracted with saturated sodium bicarbonate solution. The organic phase was washed with saturated sodium chloride solution, concentrated on a rotary evaporator, and purified by preparative HPLC. The appropriate fractions were concentrated on a rotary evaporator, and the residue was lyophilized from 1,4-dioxane. This yielded 169 mg (quantitative) of the intermediate.

LC-MS (Method 1): R _t = 1.39 min; MS (ESIpos): m/z = 658 (M+H) ⁺ .

165 mg (0.251 mmol) of this intermediate were placed in 30 ml of ethanol, and 0.35 ml of a 40% aqueous methylamine solution was added. The preparation was stirred at 50°C for 5 hours, after which the same amount of methylamine solution was added. After stirring for a further 10 hours, the preparation was concentrated under vacuum. The mixture was redistilled twice from diethyl ether, and the residue was then lyophilized from acetonitrile/water. This yielded 148 mg (89%) of this intermediate.

LC-MS (Method 6): R _t = 2.97 min; MS (ESIpos): m/z = 616 (M+H) ⁺ .

98 mg (0.15 mmol) of this precursor were dissolved in 15 ml of THF, followed by the addition of a solution of 569 mg (3.27 mmol) of disodium dithionite in 6 ml of water at room temperature. After stirring at 50°C for 8 hours, the same amount of dithionite dissolved in 1 ml of _H₂O was added. After stirring at 50°C for a further 16 hours, the preparation was cooled to room temperature and extracted with ethyl acetate. The organic phase was concentrated, and the residue was purified by preparative HPLC. The residue was lyophilized from 1,4-dioxane to yield 44.5 mg (47% of theory) of the title compound.

LC-MS (Method 1): R _t = 1.24 min; MS (ESIpos): m/z = 586 (M+H) ⁺ .

Intermediate C11

R/S-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl)-homocysteine/trifluoroacetate (1:1)

Initially, 990.0 mg (2.79 mmol) of (1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropan-1-amine was charged in 15.0 mL of dichloromethane. 828.8 mg (3.91 mmol) of sodium triacetoxyborohydride and 129.9 mg (3.21 mmol) of acetic acid were added and stirred at room temperature for 5 minutes. 698.1 mg (3.21 mmol) of 2-(trimethylsilyl)ethyl (3-oxopropyl)carbamate (Intermediate L58), dissolved in 15.0 mL of dichloromethane, was added, and the reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with ethyl acetate, and the organic phase was washed twice with saturated sodium carbonate solution and twice with saturated NaCl solution. The organic phase was dried over magnesium sulfate, and the solvent was evaporated under vacuum. The residue was purified on silica gel (eluent: dichloromethane/methanol 100:2). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 1.25 g (73% of theory) of the compound 2-(trimethylsilyl)ethyl 3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)propyl]carbamate.

LC-MS (Method 1): R _t = 1.09 min; MS (ESIpos): m/z = 556 (M+H) ⁺ .

151.4 mg (1.5 mmol) of triethylamine and 161.6 mg (1.43 mmol) of chloroacetyl chloride were added to 400.0 mg (0.65 mmol) of 2-(trimethylsilyl)ethyl 3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)propyl]carbamate. The reaction mixture was stirred at room temperature overnight. Ethyl acetate was added to the reaction mixture, and the organic phase was washed three times with water and once with saturated NaCl solution. The mixture was dried over magnesium sulfate and the solvent was evaporated under vacuum. The residue was purified using silica gel (eluent: cyclohexane/ethyl acetate 3:1). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 254.4 mg (57% of theory) of the compound 2-(trimethylsilyl)ethyl 3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(chloroacetyl)amino]propyl}carbamate.

LC-MS (Method 1): R _t = 1.49 min; MS (ESIneg): m/z = 676 (M+HCOO ^- ) ^- .

117.4 mg (0.19 mmol) of 2-(trimethylsilyl)ethyl {3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(chloroacetyl)amino]propyl}carbamate was dissolved in 10.0 ml of isopropanol, and 928.4 μl of 1 M NaOH and 50.2 mg (0.37 mmol) of DL-homocysteine were added. The reaction mixture was stirred at 50°C for 4.5 hours. Ethyl acetate was added to the reaction mixture, and the organic phase was washed with saturated sodium bicarbonate solution and saturated NaCl solution. The organic phase was dried over magnesium sulfate, and the solvent was evaporated under vacuum. The residue was purified by preparative RP-HPLC (column: Reprosil 250x40; 10µm, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum and the residue was dried under high vacuum. This gave 75.3 mg (48% of theory) of the title compound.

LC-MS (Method 1): R _t = 1.24 min; MS (ESIpos): m/z = 731 (M+H) ⁺ .

¹ H NMR (400 MHz, DMSO-d ₆ ): δ [ppm] = 0.03 (s, 9H), 0.40 (m, 1H), 0.75-0.91 (m, 11H), 1.30 (m, 1H), 1.99-2.23 (m, 2H), 2.63-2.88 (m, 4H), 3.18-3.61(m, 5H), 3.79-4.10 (m, 3H), 4.89 (d, 1H), 4.89 (d, 1H), 5.16 (d, 1H), 5.56(s, 1H), 6.82 (m, 1H), 6.91 (s, 1H), 6.97 (m, 1H), 7.13-7.38 (m, 6H), 7.49(s, 1H), 7.63(m, 1H), 8.26(s, 3H).

Intermediate C12

R/S-[(8S)-11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-8-carboxy-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl]homocysteine

The synthesis was carried out similarly to the synthesis of Intermediate C11 using methyl (2S)-4-oxo-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoate (Intermediate L57) and Intermediate C52 as starting materials.

LC-MS (Method 1): R _t = 1.18 min; MS (ESIpos): m/z = 775 (M+H) ⁺ .

Intermediate C13

9-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-4,10-dioxo-3-oxa-12-thia-5,9-diazaoctadecane-18-oic acid

90.0 mg (0.15 mmol) of intermediate C16 and 43.6 mg (0.23 mmol) of 6-(acetylsulfanyl)hexanoic acid were dissolved in 9.0 ml of methanol, and one drop of water and 73.9 mg (0.54 mmol) of potassium carbonate were added. The reaction mixture was stirred at 50°C for 4 hours and then diluted with ethyl acetate. The organic phase was washed with water/saturated NaCl solution and saturated NaCl solution, then dried over magnesium sulfate. The solvent was evaporated under vacuum, and the residue was chromatographed on silica gel (eluent: dichloromethane/methanol = 100:2). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded the title compound at 83% of theory.

LC-MS (Method 1): R _t = 1.44 min; MS (ESIpos): m/z = 701 (M+H) ⁺ .

Intermediate C14

R/S-[2-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}{3-[(tert-butoxycarbonyl)amino]propyl}amino)-2-oxoethyl]homocysteine

Initially, 100.0 mg (0.17 mmol) of tert-butyl {3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(chloroacetyl)amino]propyl}carbamate (Intermediate C16) was charged in 4.0 mL of isopropanol, and 276.5 mg (0.85 mmol) of 1 M NaOH solution and 45.9 mg (0.34 mmol) of D/L-homocysteine were added. The reaction mixture was stirred at 50°C for 1 hour. The reaction mixture was diluted with ethyl acetate. The organic phase was washed with saturated sodium bicarbonate solution and saturated NaCl solution. The mixture was dried over magnesium sulfate, and the solvent was evaporated under vacuum. The residue was purified by preparative RP-HPLC (column: Reprosil 250x40; 10µm, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum and the residue was dried under high vacuum.

This gave 92.6 mg (66% of theory) of the title compound.

LC-MS (Method 1): R _t = 1.07 min; MS (ESIpos): m/z = 688 (M+H) ⁺ .

Intermediate C15

tert-Butyl [3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino)propyl]carbamate

750.0 mg (2.11 mmol) of N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide (Intermediate C1) was dissolved in 15.0 ml of dichloromethane. 626.0 mg (2.95 mmol) of sodium triacetoxyborohydride and 139 μL (2.43 mmol) of HOAc were added, and the mixture was stirred at room temperature for 5 minutes. Then, 420.3 mg (2.43 mmol) of tert-butyl (3-oxopropyl)carbamate (synthesized according to the literature procedure, J. Med. Chem. 2003, 46, 3536) was added, and the mixture was stirred at room temperature overnight. Ethyl acetate was added to the reaction mixture, and the mixture was extracted twice with saturated sodium carbonate solution. The organic phase was washed with saturated NaCl solution and dried over magnesium sulfate. The solvent was evaporated under vacuum, and the residue was chromatographed on silica gel (eluent: cyclohexane/ethyl acetate = 4:1). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 881.0 mg (82% of theory) of the title compound.

LC-MS (method 1): R _t = 1.07 min; MS (ESIpos): m/z = 513 [M+H] ⁺ .

Intermediate C16

tert-Butyl {3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(chloroacetyl)amino]propyl}carbamate

373.4 mg (0.73 mmol) of tert-butyl 3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino)propyl]carbamate (Intermediate C15) were initially charged in 5.0 ml of dichloromethane, and 169.5 mg (1.68 mmol) of triethylamine and 181.0 mg (1.60 mmol) of chloroacetyl chloride were added. The reaction mixture was stirred at room temperature overnight, then ethyl acetate was added and repeatedly extracted with water. The organic phase was washed with saturated NaCl solution and dried over magnesium sulfate. The solvent was evaporated under vacuum, and the residue was chromatographed on silica gel (eluent: dichloromethane/methanol = 100:0.5). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 336.0 mg (75% of theory) of the title compound.

LC-MS (Method 1): R _t = 1.48 min; MS (ESIpos): m/z = 589 [M+H] ⁺ .

Intermediate C17

9-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-4,10-dioxo-3,15,18,21,24-pentaoxa-12-thia-5,9-diazaheptacosanoic acid

Initially, 50.0 mg (0.09 mmol) of tert-butyl {3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(chloroacetyl)amino]propyl}carbamate (Intermediate C16) was charged in 2.0 ml of DMF, and 69.1 mg (0.21 mmol) of cesium carbonate and 28.8 mg (0.10 mmol) of 1-sulfanyl-3,6,9,12-tetraoxapentadecan-15-oic acid were added. The mixture was stirred at 50°C overnight. Water was added to the reaction mixture and purified directly by preparative RP-HPLC (column: Reprosil 250x30; 10µm, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 25.1 mg (35% of theory) of the title compound.

LC-MS (Method 1): R _t = 1.42 min; MS (ESIpos): m/z = 835 [M+H] ⁺ .

Intermediate C18

tert-Butyl [22-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4,21-dioxo-7,10,13,16-tetraoxa-19-thia-3,22-diazapentacosan-25-yl]carbamate

21.0 mg (0.03 mmol) of 9-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-4,10-dioxo-3,15,18,21,24-pentaoxa-12-thia-5,9-diazaheptacosanoic acid (Intermediate C17) and 5.8 mg (0.0.3 mmol) of 1-(2-aminoethyl)-1H-pyrrole-2,5-dione hydrochloride (1:1) were initially charged in 1.0 mL of acetonitrile, and 26.1 mg (0.20 mmol) of N , N -diisopropylethylamine and 20.9 mg (0.03 mmol) of T3P (50% in ethyl acetate) were added. The mixture was stirred at room temperature overnight. The reaction mixture was purified directly by preparative RP-HPLC (column: Reprosil 250x30; 10µ, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 19.7 mg (79% of theory) of the title compound.

LC-MS (Method 1): R _t = 1.42 min; MS (ESIpos): m/z = 835 [M+H] ⁺ .

Intermediate C19

tert-Butyl (13-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-10-thia-7,13-diaza-2-silahexadec-16-yl)carbamate

Initially, 58.5 mg (0.10 mmol) of tert-butyl {3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(chloroacetyl)amino]propyl}carbamate (Intermediate C16) was charged in 2.0 mL of DMF. 44.0 mg (0.20 mmol) of 2-(trimethylsilyl)ethyl (2-sulfanylethyl)carbamate (Intermediate L39) and 64.7 mg (0.20 mmol) of cesium carbonate were added. The mixture was stirred at 50°C for 4 hours. This formulation was repeated using 46.6 mg (0.079 mmol) of Intermediate C16. The two reaction mixtures were combined and directly purified by preparative RP-HPLC (column: Reprosil 250x30; 10µm, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum and the residue was dried under high vacuum. This gave 98.0 mg (71% of theory) of the title compound.

LC-MS (method 1): R _t = 1.62 min; MS (ESIpos): m/z = 774 [M+H] ⁺ .

Intermediate C20

trifluoroacetic acid/tert-butyl [3-({[(2-aminoethyl)sulfanyl]acetyl}{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino)propyl]carbamate

98.0 mg (0.13 mmol) of tert-butyl (13-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-10-thia-7,13-diaza-2-silahexadec-16-yl)carbamate (Intermediate C19) were initially charged in 2.0 ml of DMF/tert-butanol (9:1), and 96.2 mg (0.63 mmol) of CsF were added. The mixture was stirred at 90°C for 16 hours. The reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 250x30; 10µm, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was lyophilized. This gave 57.1 mg (61% of theory) of the title compound, which also contained the corresponding sulfoxide.

LC-MS (Method 1): R _t = 1.08 min; MS (ESIpos): m/z = 630 [M+H] ⁺ .

Intermediate C21

tert-Butyl [38-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,31,37-trioxo-7,10,13,16,19,22,25,28-octaoxa-35-thia-4,32,38-triazatetriacontan-41-yl]carbamate

57.1 mg (0.08 mmol) of trifluoroacetic acid/tert-butyl [3-({[(2-aminoethyl)sulfanyl]acetyl}{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino)propyl]carbamate (Intermediate C20) was initially charged in 3.0 ml of DMF, and 53.0 mg (0.08 mmol) of 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-{27-[(2,5-dioxopyrrolidin-1-yl)oxy]-27-oxo-3,6,9,12,15,18,21,24-octaoxaheptacosan-1-yl}propanamide and 15.5 mg (0.15 mmol) of triethylamine were added. The mixture was stirred at room temperature for 16 hours. The reaction mixture was purified directly by preparative RP-HPLC (column: Reprosil 250x30; 10µ, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum and the residue was lyophilized. This yielded 49.7 mg (49% of theory) of the title compound.

LC-MS (method 1): R _t = 1.34 min; MS (ESIpos): m/z = 1204 [M+H] ⁺ .

Intermediate C22

tert-Butyl [38-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-35-oxido-3,31,37-trioxo-7,10,13,16,19,22,25,28-octaoxa-35λ4-thia-4,32,38-triazatetriacontan-41-yl]carbamate

The title compound was formed as a by-product in the synthesis of intermediate C21. This gave 15.5 mg (15% of theory) of the title compound.

LC-MS (Method 1): R _t = 1.25 min; MS (ESIpos): m/z = 1220 [M+H] ⁺ .

Intermediate C23

tert-Butyl 3-amino-4-{[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]methyl}pyrrolidine-1-carboxylate

mixture of stereoisomers

Initially, 411.2 mg (1.15 mmol) of tert-butyl 3-formyl-4-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)pyrrolidine-1-carboxylate (Intermediate L28) and 339.7 mg (0.96 mmol) of N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide (Intermediate C1) were charged in 6.0 mL of dichloromethane, 68.9 mg (1.15 mmol) of HOAc was added, and the mixture was stirred at room temperature for 1 hour. 405.2 mg (1.91 mmol) of sodium triacetoxyborohydride was added, and the mixture was stirred at room temperature for 2 hours. The solvent was evaporated under vacuum, and ethyl acetate and water were added to the residue. The aqueous phase was extracted three times with ethyl acetate. The combined organic phases were washed once with saturated NaCl solution and dried over magnesium sulfate. The solvent was evaporated under vacuum, and the residue was purified using Biotage Isolera (silica gel, 50 g SNAP column, flow rate 40 ml/min, petroleum ether/ethyl acetate). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 541.5 mg (81% of theory) of the compound tert-butyl 3-[({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino)methyl]-4-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)pyrrolidine-1-carboxylate.

LC-MS (method 1): R _t = 1.24 and 1.29 min; MS (ESIpos): m/z = 698 [M+H] ⁺ .

541.5 mg (0.78 mmol) of tert-butyl 3-[({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino)methyl]-4-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)pyrrolidine-1-carboxylate was dissolved in 13.0 ml of dichloromethane, and 180.6 mg (1.78 mmol) of triethylamine was added. The reaction solution was cooled to 0°C, 233.1 mg (1.71 mmol) of acetoxyacetyl chloride was added, and the mixture was stirred at room temperature for 16 hours. An additional 180.6 mg (1.78 mmol) of triethylamine and 233.1 mg (1.71 mmol) of acetoxyacetyl chloride were added, and the mixture was stirred at room temperature for an additional 80 hours. The solvent was evaporated under vacuum, and the residue was partitioned between water and ethyl acetate. The aqueous phase was extracted three times with ethyl acetate. The combined organic phases were washed once with saturated NaCl solution and dried over magnesium sulfate. The solvent was evaporated under vacuum, and the residue was purified using a Biotage Isolera (silica gel, 50 g SNAP column, flow rate 40 ml/min, petroleum ether/ethyl acetate). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 529.2 mg (86% of theory) of the compound tert-butyl 3-{[(acetoxyacetyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino]methyl}-4-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)pyrrolidine-1-carboxylate.

LC-MS (method 1): R _t = 1.53 and 1.56 min; MS (ESIpos): m/z = 798 [M+H] ⁺ .

529.2 mg (0.66 mmol) of tert-butyl 3-{[(acetoxyacetyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino]methyl}-4-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)pyrrolidine-1-carboxylate was initially charged in 10.0 mL of DMF/tert-butanol (9:1), and 503.7 mg (3.32 mmol) of CsF was added. The reaction mixture was stirred at 90°C for 16 hours. The reaction mixture was partitioned between water and ethyl acetate. The aqueous phase was extracted three times with ethyl acetate. The combined organic phases were washed once with saturated NaCl solution and dried over magnesium sulfate. The solvent was evaporated under vacuum, and the residue was purified using Biotage Isolera (silica gel, 50 g SNAP column, flow rate 25 mL/min, dichloromethane/methanol). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 172.4 mg (40% of theory) of the compound tert-butyl 3-{[(acetoxyacetyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino]methyl}-4-aminopyrrolidine-1-carboxylate.

LC-MS (method 1): R _t = 1.05 and 1.35 min; MS (ESIpos): m/z = 654 [M+H] ⁺ .

Initially, 172.4 mg (0.26 mmol) of tert-butyl 3-{[(acetoxyacetyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino]methyl}-4-aminopyrrolidine-1-carboxylate was charged in 4.5 mL of methanol/water (2:1). 80.2 mg (0.58 mmol) of potassium carbonate was added and stirred at room temperature for 16 hours. The reaction mixture was partitioned between water and ethyl acetate. The aqueous phase was extracted three times with ethyl acetate. The combined organic phases were washed once with saturated NaCl solution and dried over magnesium sulfate. The solvent was evaporated under vacuum, and the residue was purified by preparative RP-HPLC (column: Reprosil 250x30; 10µm, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 116.0 mg (72% of theory) of the title compound.

LC-MS (method 1): R _t = 1.01 min and 1.03 min; MS (ESIpos): m/z = 612 [M+H] ⁺ .

Intermediate C24

Trifluoroacetic acid/tert-butyl 3-(aminomethyl)-4-{[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]methyl}pyrrolidine-1-carboxylate (1:1)

26.8 mg of N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide (Intermediate C1) was dissolved in 3.0 ml of dichloromethane, and 5.2 mg (0.09 mmol) of HOAc and 22.4 mg (0.11 mmol) of sodium triacetoxyborohydride were added, followed by stirring at room temperature for 5 minutes. 62.4 mg (0.09 mmol) of tert-butyl 3-formyl-4-[({[2-(trimethylsilyl)ethoxy]carbonyl}amino)methyl]pyrrolidine-1-carboxylate (Intermediate L29) was added, and the mixture was stirred at room temperature overnight. The solvent was evaporated under vacuum, and the residue was purified by preparative RP-HPLC (column: Reprosil 250x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 57.6 mg (91% of theory) of the compound trifluoroacetic acid/tert-butyl 3-[({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino)methyl]-4-[({[2-(trimethylsilyl)ethoxy]carbonyl}amino)methyl]pyrrolidine-1-carboxylate.

LC-MS (method 1): R _t = 1.25 and 1.27 min; MS (ESIpos): m/z = 712 [M+H] ⁺ .

77.0 mg (0.11 mmol) of tert-butyl 3-[({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino)methyl]-4-[({[2-(trimethylsilyl)ethoxy]carbonyl}amino)methyl]pyrrolidine-1-carboxylate were initially charged in 1.5 ml of dichloromethane, and 21.9 mg (0.22 mmol) of triethylamine were added. 29.5 mg (0.22 mmol) of acetoxyacetyl chloride were then added at 0°C, and the reaction mixture was stirred at room temperature overnight. The solvent was evaporated under vacuum, and the residue was taken up in ethyl acetate. The organic phase was washed once with water, once with saturated sodium bicarbonate solution, and once with saturated NaCl solution. After drying over magnesium sulfate, the solvent was evaporated under vacuum. The preparation was repeated with 77.0 mg (0.11 mmol) of tert-butyl 3-[({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino)methyl]-4-[({[2-(trimethylsilyl)ethoxy]carbonyl}amino)methyl]pyrrolidine-1-carboxylate. The combined residues were purified on silica gel (eluent: cyclohexane/ethyl acetate = 2:1). This gave 171.1 mg (85% of theory) of the compound tert-butyl 3-{[(acetoxyacetyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino]methyl}-4-[({[2-(trimethylsilyl)ethoxy]carbonyl}amino)methyl]pyrrolidine-1-carboxylate.

LC-MS (method 1): R _t = 1.56 and 1.57 min; MS (ESIpos): m/z = 812 [M+H] ⁺ .

Dissolve 30.0 mg (0.04 mmol) of tert-butyl 3-{[(acetoxyacetyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino]methyl}-4-[({[2-(trimethylsilyl)ethoxy]carbonyl}amino)methyl]pyrrolidine-1-carboxylate in 0.5 ml of TBAF solution (1 M in THF). Stir overnight at room temperature. The solvent is evaporated under vacuum, and the residue is purified by preparative RP-HPLC (column: Reprosil 250x30; 10µm, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent is evaporated under vacuum, and the residue is dried under high vacuum. This yields 25.0 mg (92% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.98 min; MS (ESIpos): m/z = 626 [M+H] ⁺ .

Intermediate C25

4-{[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]methyl}-1-(tert-butoxycarbonyl)pyrrolidine-3-carboxylic acid

Initially, 171.4 mg (0.48 mmol) of N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide (Intermediate C1) was charged in 4.0 ml of dichloromethane, and 248.5 mg (0.72 mmol) of tert-butyl 3-({[tert-butyl(dimethyl)silyl]oxy}methyl)-4-formylpyrrolidine-1-carboxylate (Intermediate L30) and 34.8 mg (0.58 mmol) of HOAc were added. The reaction mixture was stirred at room temperature for 1 hour. 204.4 mg (0.97 mmol) of sodium triacetoxyborohydride was added, and the mixture was stirred at room temperature for 60 hours. The solvent was removed under vacuum, and the residue was purified using a Biotage Isolera (silica gel, 25 g SNAP column, flow rate 25 ml/min, petroleum ether/ethyl acetate). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 267.0 mg (77% of theory) of tert-butyl 3-[({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino)methyl]-4-({[tert-butyl(dimethyl)silyl]oxy}methyl)pyrrolidine-1-carboxylate.

LC-MS (Method 1): R _t = 1.49 min; MS (ESIpos): m/z = 683 [M+H] ⁺ .

267.0 mg (0.39 mmol) of tert-butyl 3-[({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino)methyl]-4-({[tert-butyl(dimethyl)silyl]oxy}methyl)pyrrolidine-1-carboxylate was dissolved in 5.0 ml of dichloromethane, 91.0 mg (0.90 mmol) of triethylamine was added, and the mixture was cooled to 0°C. 117.4 mg (0.86 mmol) of acetoxyacetyl chloride was added, and the mixture was stirred at room temperature for 16 hours. An additional 593.4 mg (5.87 mmol) of triethylamine and 427.0 mg (3.13 mmol) of acetoxyacetyl chloride were added, and the mixture was stirred at room temperature for an additional 10 hours. The solvent was evaporated under vacuum, and the residue was purified by preparative RP-HPLC (column: Reprosil 250x30; 10µ, flow rate: 50 ml/min, MeCN/water). The solvent was then evaporated under vacuum, and the residue was dried under high vacuum. This yielded 216.3 mg (71% of theory) of tert-butyl 3-{[(acetoxyacetyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino]methyl}-4-({[tert-butyl(dimethyl)silyl]oxy}methyl)pyrrolidine-1-carboxylate.

LC-MS (method 1): R _t = 1.70 and 1.72 min; MS (ESIpos): m/z = 783 [M+H] ⁺ .

216.3 mg (0.28 mmol) of tert-butyl 3-{[(acetoxyacetyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino]methyl}-4-({[tert-butyl(dimethyl)silyl]oxy}methyl)pyrrolidine-1-carboxylate was initially charged in 4.0 mL of THF, and 16.6 mg (0.28 mmol) of HOAc and 361.1 mg (1.38 mmol) of a 1 M TBAF solution in THF were added. The reaction solution was stirred at room temperature for 4 hours. The solvent was evaporated under vacuum, and the residue was purified by preparative RP-HPLC (column: Reprosil 250x30; 10µm, flow rate: 50 ml/min, MeCN/water). The solvent was then evaporated under vacuum, and the residue was dried under high vacuum. This gave 94.0 mg (51% of theory) of the compound tert-butyl 3-{[(acetoxyacetyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino]methyl}-4-(hydroxymethyl)pyrrolidine-1-carboxylate.

LC-MS (Method 1): R _t = 1.34 min; MS (ESIpos): m/z = 669 [M+H] ⁺ .

52.0 mg (0.08 mmol) of tert-butyl 3-{[(acetoxyacetyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino]methyl}-4-(hydroxymethyl)pyrrolidine-1-carboxylate was initially charged in 4.0 mL of PBS buffer/acetonitrile (1:1), and 1.2 mg (0.01 mmol) of TEMPO was added. Then, 14.1 mg (0.16 mmol) of sodium chlorite and 115.8 μL (0.16 mmol) of 10% sodium hypochlorite solution in 1.0 mL of water were added simultaneously. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was poured into 10% sodium sulfite solution, and ethyl acetate was added. The aqueous phase was extracted three times with ethyl acetate, and the combined organic phases were washed once with saturated NaCl solution and dried over magnesium sulfate. The solvent was evaporated under vacuum and the residue was used in the next synthetic step without further purification.

LC-MS (Method 1): R _t = 1.34 min; MS (ESIpos): m/z = 683 [M+H] ⁺ .

Initially, 103.0 mg (0.15 mmol) of 4-{[(acetoxyacetyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino]methyl}-1-(tert-butoxycarbonyl)pyrrolidine-3-carboxylic acid was charged in 4.5 mL of methanol/water (2:1). 45.9 mg (0.33 mmol) of potassium carbonate was added and stirred at room temperature for 3 hours. The reaction mixture was partitioned between water and ethyl acetate. The aqueous phase was extracted three times with ethyl acetate, and the combined organic phases were washed once with saturated NaCl solution and dried over magnesium sulfate. The solvent was evaporated under vacuum, and the title compound was used in the next synthetic step without further purification.

LC-MS (Method 1): R _t = 1.35 min; MS (ESIpos): m/z = 641 [M+H] ⁺ .

Intermediate C26

tert-Butyl [3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino)-2-({[tert-butyl(dimethyl)silyl]oxy}methyl)propyl]carbamate

590 mg (1.69 mmol) of sodium triacetoxyborohydride and 155 μl (2.70 mmol, 162 mg) of acetic acid were initially charged in 30 ml of dichloromethane, and the mixture was stirred at room temperature for 30 minutes. Then, 600 mg (1.687 mmol) of (1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropan-1-amine (from trifluoroacetic acid/(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropan-1-amine (1:1) by extraction with 1N aqueous sodium hydroxide solution) and 750 mg (2.362 mmol) of tert-butyl (3-{[tert-butyl(dimethyl)silyl]oxy}-2-formylpropyl)carbamate dissolved in 40 ml of dichloromethane were added dropwise. The mixture was stirred at room temperature for 2 hours. Ethyl acetate was then added, the mixture was washed with saturated sodium carbonate solution, and the organic phase was concentrated. The residue was separated by preparative HPLC (eluent: ACN/water gradient). This gave 510 mg (46% of theory) of the target compound as a mixture of diastereomers.

Isomer 1:

LC-MS (Method 1): R _t = 1.36 min (51%); MS (EIpos): m/z = 657 [M+H] ⁺ .

Isomer 2:

LC-MS (Method 1): R _t = 1.41 min (49%); MS (EIpos): m/z = 657 [M+H] ⁺ .

Intermediate C27

2-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}{3-[(tert-butoxycarbonyl)amino]-2-({[tert-butyl(dimethyl)silyl]oxy}methyl)propyl}amino)-2-oxoethyl acetate

510 mg (0.776 mmol) of tert-butyl [3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino)-2-({[tert-butyl(dimethyl)silyl]oxy}methyl)propyl]carbamate were initially charged in 30 ml of dichloromethane, and 181 mg (249 μl, 1.786 mmol) of triethylamine and 219 mg (1.553 mmol) of 2-chloro-2-oxoethyl acetate were added at room temperature. The reaction mixture was stirred at room temperature for 2 hours and then washed with saturated sodium bicarbonate solution. The organic phase was dried over sodium sulfate and concentrated on a rotary evaporator. The residue was separated by preparative HPLC (eluent: ACN/water, gradient). This gave 290 mg (49% of theory) of the target compound as a mixture of epimers.

Isomer 1:

LC-MS (Method 1): R _t = 1.70 min; MS (EIpos): m/z = 757 [M+H] ⁺ .

Isomer 2:

LC-MS (Method 1): R _t = 1.72 min; MS (EIpos): m/z = 757 [M+H] ⁺ .

Intermediate C28

2-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}{3-[(tert-butoxycarbonyl)amino]-2-(hydroxymethyl)propyl}amino)-2-oxoethyl acetate

285 mg (0.376 mmol) of 2-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}{3-[(tert-butoxycarbonyl)amino]-2-({[tert-butyl(dimethyl)silyl]oxy}methyl)propyl}amino)-2-oxoethyl acetate was dissolved in 5 ml of THF. 452 μl (0.452 mmol) of a 1 M solution of tetra-n-butylammonium fluoride in THF were added, and the reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was separated by preparative HPLC (eluent: ACN/water, gradient) and lyophilized. This yielded 214 mg (81% of theory, purity according to LC/MS = 92%) of the target compound as a mixture of epimers.

Isomer 1:

LC-MS (Method 1): R _t = 1.37 min; MS (EIpos): m/z = 643 [M+H] ⁺ .

Isomer 2:

LC-MS (Method 1): R _t = 1.40 min; MS (EIpos): m/z = 643 [M+H] ⁺ .

Intermediate C29

2-([3-(Acetylsulfanyl)-2-{[(tert-butoxycarbonyl)amino]methyl}propyl]{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino)-2-oxoethyl acetate

210 mg (0.301 mmol) of 2-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}{3-[(tert-butoxycarbonyl)amino]-2-(hydroxymethyl)propyl}amino)-2-oxoethyl acetate was initially charged in 8 ml of pure THF. 178 mg (1.503 mmol, 109 μl) of thionyl chloride dissolved in 8 ml of pure THF was added dropwise at room temperature and stirred for 40 minutes. The reaction mixture was concentrated on a rotary evaporator and dried under high vacuum. The residue was taken up in 16 ml of pure DMF, 172 mg (1.503 mmol) of potassium thioacetate and 133 mg (0.361 mmol) of tetra-n-butylammonium iodide were added, and the mixture was stirred at 90°C for 2 hours. After cooling, water was added, and the mixture was extracted with ethyl acetate. The organic phase was concentrated on a rotary evaporator, and the residue was separated by preparative HPLC (eluent: ACN/water, gradient) and lyophilized. This gave 155 mg (69% of theory, purity according to LC/MS = 94%) of the target compound as a mixture of epimers.

Isomer 1:

LC-MS (Method 1): R _t = 1.50 min; MS (EIpos): m/z = 701 [M+H] ⁺ .

Isomer 2:

LC-MS (Method 1): R _t = 1.51 min; MS (EIpos): m/z = 701 [M+H] ⁺ .

Intermediate C30

Di-tert-butyl [disulfanediylbis(2-{[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]methyl}propane-3,1-diyl)]biscarbamate

1.220 g (1.010 mmol, purity according to LC/MS = 58%) of 2-([3-(acetylsulfanyl)-2-{[(tert-butoxycarbonyl)amino]methyl}propyl]{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino)-2-oxoethyl acetate were initially charged in 30 ml of THF and 30 ml of methanol, 10 ml of 1 N aqueous sodium hydroxide solution were added, and the mixture was stirred at room temperature for 2 hours. Water was added to the reaction mixture, and the mixture was extracted with dichloromethane. The organic phase was dried over sodium sulfate and concentrated on a rotary evaporator. The residue was purified by preparative HPLC (eluent: ACN/water, gradient). This gave 390 mg (54% of theory, purity according to LC/MS = 86%) of the target compound as a mixture of diastereomers.

Isomers:

LC-MS (Method 1): R _t = 1.81 min; MS (EIpos): m/z = 1232 [M+H] ⁺ .

Intermediate C31

tert-Butyl 3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-2-(sulfanylmethyl)propyl}carbamate

390 mg (0.272 mmol, purity = 86% according to LC/MS) of di-tert-butyl disulfanediylbis(2-{[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]methyl}propane-3,1-diyl)]biscarbamate were placed in 20 ml of 1,4-dioxane and 10 ml of PBS buffer, and 234 mg (0.817 mmol) of 3,3',3''-phosphanetriyltripropionic acid hydrochloride (1:1) were added. The mixture was stirred at room temperature for 16 hours. The reaction mixture was then concentrated on a rotary evaporator and stirred with dichloromethane, and the filtrate was concentrated and dried under high vacuum. The residue was dissolved in 8 ml of isopropanol and purified by chiral chromatography (column: 250 x 30 mm, packed with Daicel Chiralpak AZ-H, eluent: isohexane/isopropanol = 90:10). This yielded two fractions of the target compound. Fraction 1 contained 181.2 mg (50% of theory) of isomer 1, and fraction 2 yielded 90.2 mg (25% of theory) of isomer 2.

Isomer 1:

Chiral HPLC (column: 250 x 4.6 mm, packed with Diacel Chiralpak AZ-H, eluent: isohexane/ethanol 90:10): R _t = 6.98 min.

LC-MS (Method 1): R _t = 1.47 min; MS (EIpos): m/z = 617 [M+H] ⁺ .

Isomer 2:

Chiral HPLC (column: 250 x 4.6 mm, packed with Diacel Chiralpak AZ-H, eluent: isohexane/ethanol 90:10): R _t = 9.39 min.

LC-MS (Method 1): R _t = 1.47 min; MS (EIpos): m/z = 617 [M+H] ⁺ .

Intermediate C32

N-[3-Amino-2-(sulfanylmethyl)propyl]-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide hydrochloride (1:1) (Isomer 1)

123 mg (199.42 μmol) of tert-butyl {3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-2-(sulfanylmethyl)propyl}carbamate (Isomer 1) was dissolved in 2 ml of THF and stirred with 10 ml of semiconcentrated hydrochloric acid at room temperature for 1 hour. The reaction solution was degassed under argon and then lyophilized. This yielded 108 mg (98% of theory) of the target compound.

Isomer 1

LC-MS (Method 1): R _t = 0.95 min; MS (EIpos): m/z = 517 [M+H] ⁺ .

Intermediate C33

N-[3-Amino-2-(sulfanylmethyl)propyl]-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide hydrochloride (1:1) (Isomer 2)

123 mg (199.42 μmol) of tert-butyl {3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-2-(sulfanylmethyl)propyl}carbamate (Isomer 2) was dissolved in 2 ml of THF and stirred with 10 ml of semi-concentrated hydrochloric acid at room temperature for 1 hour. The reaction solution was degassed under argon and then lyophilized. This yielded 58 mg (63% of theory, purity according to LC/MS = 91%) of the target compound.

Isomer 2

LC-MS (method 1): R _t = 0.97 min; MS (ESIpos): m/z = 517 [M+H] ⁺ .

Intermediate C34

tert-Butyl [3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino)propyl]carbamate

3.790 g (10.02 mmol) of (1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropan-1-amine (obtained from trifluoroacetic acid/(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropan-1-amine (1:1) by extraction with 1N aqueous sodium hydroxide solution), 3.186 g (15.04 mmol) of sodium triacetoxyborohydride, and 690 μl (12.03 mmol, 722 mg) were initially charged to 100 ml of dichloromethane. The mixture was stirred at room temperature for 5 minutes.

4.687 g (27.06 mmol) of tert-butyl (3-oxopropyl)carbamate was then added, and the mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with dichloromethane and washed with saturated sodium bicarbonate solution. The organic phase was dried over sodium sulfate and concentrated on a rotary evaporator. The residue was purified by chromatography on silica gel (eluent: dichloromethane/ethyl acetate, gradient = 4:1 → 1:1). This yielded 2.57 g (48% of theory, purity according to LC/MS = 96%) of the target compound.

LC-MS (Method 1): R _t = 1.00 min; MS (EIpos): m/z = 513 [M+H] ⁺ .

Intermediate C35

tert-Butyl {3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(4-nitrobenzoyl)amino]propyl}carbamate

200 mg (0.38 mmol) of tert-butyl [3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino)propyl]carbamate were initially charged in 9 ml of pure dichloromethane, and 120 μl (0.86 mmol, 87 mg) of triethylamine were added at room temperature. 83 mg (0.45 mmol) of 4-nitrobenzoyl chloride dissolved in 1 ml of pure dichloromethane were added dropwise at room temperature, and the mixture was stirred at room temperature for 1 hour. Water was added, and the mixture was concentrated on a rotary evaporator. The residue was purified by preparative HPLC (eluent: ACN/water + 0.1% TFA, gradient) and dried. This gave 181 mg (73% of theory) of the target compound.

LC-MS (Method 1): R _t = 1.47 min; MS (EIpos): m/z = 662 [M+H] ⁺ .

Intermediate C36

tert-Butyl {3-[(4-aminobenzoyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino]propyl}carbamate

Initially, 170 mg (0.26 mmol) of tert-butyl {3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(4-nitrobenzoyl)amino]propyl}carbamate was charged in 10 ml of acetic acid. 143 mg (2.57 mmol) of iron powder was added and stirred at 50°C for 16 hours. After cooling, water was added and the mixture was extracted with ethyl acetate. The organic phase was dried over sodium sulfate and concentrated on a rotary evaporator. The residue was dried under HV. This yielded 154 mg (77% of theory, purity according to LC/MS = 82%) of the target compound.

LC-MS (Method 5): R _t = 4.73 min; MS (EIpos): m/z = 632 [M+H] ⁺ .

Intermediate C37

N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecanoyl]-L-valyl-N-[4-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}{3-[(tert-butoxycarbonyl)amino]propyl}carbamoyl)phenyl]-L-alaninamide

38.6 mg (0.05 mmol, LC/MS purity = 82%) of tert-butyl {3-[(4-aminobenzoyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino]propyl}carbamate was dissolved in pure DMF, and 24.8 mg (0.06 mmol) of HATU and 13.0 mg (0.10 mmol) of N , N -diisopropylethylamine were added. The mixture was stirred at room temperature for 5 minutes, then 63 mg (0.06 mmol) of N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecanoyl]-L-valyl-L-alanine was added and stirred at room temperature for 3 hours. 7.5 mg (0.06 mmol) of 3H-[1,2,3]triazolo[4,5-b]pyridin-3-ol (HOAt) was added and stirred for 16 hours. 19.1 mg (0.05 mmol) of HATU was added and the mixture was stirred at 50°C for 2 hours. After cooling, the reaction mixture was directly purified by preparative HPLC (eluent: ACN/water + 0.1% TFA, gradient). This yielded 6.5 mg (9% of theory, purity according to LC/MS = 83%) of the target compound.

LC-MS (Method 2): R _t = 7.89 min; MS (EIpos): m/z = 1200.6 [M+H] ⁺ .

Intermediate C38

2-[3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino)propyl]-1H-isoindole-1,3(2H)-dione

300.0 mg (0.84 mmol) of 2-[3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino)propyl]-1H-isoindole-1,3(2H)-dione (Intermediate C1) were initially charged in 4.0 ml of dichloromethane, 58.3 mg (0.97 mmol) of HOAc and 250.4 mg (1.18 mmol) of sodium triacetoxyborohydride were added, and the mixture was stirred at room temperature for 5 minutes. 197.2 mg (0.97 mmol) of 3-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)propanal was added. The reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with ethyl acetate, and the organic phase was washed twice with saturated sodium carbonate solution and once with saturated NaCl solution. After drying over magnesium sulfate, the solvent was evaporated under vacuum and the residue was purified on silica gel (eluent: ethyl acetate/cyclohexane 1:5). The solvent was evaporated under vacuum and the residue was dried under high vacuum. This yielded 333.3 mg (70%) of the title compound.

LC-MS (method 1): R _t = 1.05 min; MS (ESIpos): m/z = 543 [M+H] ⁺ .

Intermediate C39

2-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}[3-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)propyl]amino)-2-oxoethyl acetate

332.3 mg (0.61 mmol) of 2-[3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino)propyl]-1H-isoindole-1,3(2H)-dione (Intermediate C38) were initially charged in 8.0 ml of dichloromethane, and 142.5 mg (1.35 mmol) of triethylamine were added. 184.0 mg (1.35 mmol) of acetoxyacetyl chloride were added at 0°C, and the reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with ethyl acetate, and the organic phase was washed twice with saturated sodium bicarbonate solution and once with saturated NaCl solution. After drying over magnesium sulfate, the solvent was evaporated under vacuum, and the residue was purified on silica gel (eluent: ethyl acetate/cyclohexane 1:3). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 367.1 mg (63%) of the title compound.

LC-MS (Method 1): R _t = 1.42 min; MS (ESIpos): m/z = 643 [M+H] ⁺ .

Intermediate C40

N-(3-Aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide

583.1 mg (0.91 mmol) of 2-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}[3-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)propyl]amino)-2-oxoethyl acetate (Intermediate C39) was initially charged in 15.0 ml of ethanol, and 1.41 g (18.15 mmol) of methylamine (40% in water) was added. The reaction mixture was stirred at 50°C overnight. The solvent was evaporated under vacuum, and the residue was co-distilled three times with toluene. The residue was purified on silica gel (eluent: dichloromethane/methanol = 100:5). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 324.9 mg (73%) of the title compound.

LC-MS (Method 1): R _t = 0.97 min; MS (ESIpos): m/z = 471 [M+H] ⁺ .

Intermediate C41

Trifluoroacetic acid/L-valyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-L-alaninamide (1:1)

50.0 mg (0.11 mol) of N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide (Intermediate C40) and 30.4 mg (0.11 mmol) of N-(tert-butoxycarbonyl)-L-alanine 2,5-dioxopyrrolidin-1-yl ester were initially charged in 2.0 mL of DMF, and 32.2 mg (0.32 mmol) of 4-methylmorpholine were added. The reaction mixture was stirred at room temperature overnight. 19.1 mg (0.32 mmol) of HOAc was added, and the reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 250x30; 10µm, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This resulted in 38.0 mg (56%) of the compound [(2S)-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}amino)-1-oxopropan-2-yl]carbamic acid tert-butyl ester.

LC-MS (Method 1): R _t = 1.26 min; MS (ESIpos): m/z = 642 [M+H] ⁺ .

Initially, 33.6 mg (0.05 mmol) of tert-butyl [(2S)-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}amino)-1-oxopropan-2-yl]carbamate was added to 3.0 ml of dichloromethane. 119.4 mg (1.05 mmol) of TFA was added to the reaction mixture and stirred at room temperature overnight. The solvent was evaporated under vacuum, and the residue was directly purified by preparative RP-HPLC (column: Reprosil 250x30; 10µm, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This resulted in 32.8 mg (96%) of the compound trifluoroacetic acid/N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-L-alaninamide (1:1).

LC-MS (method 1): R _t = 0.93 min; MS (ESIpos): m/z = 542 [M+H] ⁺ .

29.5 mg (0.05 mmol) of trifluoroacetic acid/N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-L-alaninamide (1:1) and 14.1 mg (0.05 mmol) of N-(tert-butoxycarbonyl)-L-valine 2,5-dioxopyrrolidin-1-yl ester were initially charged in 1.0 ml of DMF, and 18.2 mg (0.18 mmol) of 4-methylmorpholine were added. The reaction mixture was stirred at room temperature overnight and directly purified by preparative RP-HPLC (column: Reprosil 250x30; 10µm, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This resulted in 23.1 mg (69%) of the compound N-(tert-butoxycarbonyl)-L-valyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-L-alaninamide.

LC-MS (Method 1): R _t = 1.30 min; MS (ESIpos): m/z = 741 [M+H] ⁺ .

19.4 mg (0.03 mmol) of N-(tert-butoxycarbonyl)-L-valyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(glycolyl)amino]propyl}-L-alaninamide was dissolved in 1.5 ml of dichloromethane, and 59.7 mg (0.52 mmol) of TFA was added. The reaction mixture was stirred at room temperature overnight. 119.4 mg (1.04 mmol) of TFA was added, and the mixture was stirred at room temperature again overnight. The solvent was evaporated under vacuum, and the residue was directly purified by preparative RP-HPLC (column: Reprosil 250x30; 10µm, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 19.2 mg (97%) of the title compound.

LC-MS (Method 1): R _t = 0.96 min; MS (ESIpos): m/z = 641 [M+H] ⁺ .

Intermediate C42

2,5-Difluorobenzenediazonium tetrafluoroborate

Initially, 3.00 g (21.16 mmol, 2.68 ml) of boron trifluoride-diethyl ether complex was charged, and 1.37 g (10.58 mmol) of 2,5-difluoroaniline dissolved in 27 ml of pure THF was slowly added dropwise at 0°C. At -10°C, a solution of 1.61 g (13.75 mmol, 1.85 ml) of isoamyl nitrite in 3 ml of pure THF was added dropwise, and stirring was continued at the same temperature for 30 minutes. 15 ml of diethyl ether was added, and the precipitated diazonium salt was filtered off, washed with a small amount of diethyl ether, and dried under high vacuum. This yielded 2.27 g of the target compound (94% of theory).

LC-MS (Method 6): R _t = 0.24 min; MS (ESIpos): m/z = 141 [M] ⁺ .

¹ H NMR (400 MHz, DMSO-d ₆ ): δ [ppm] = 8.11-8.17 (m, 1H), 8.36-8.43 (m, 1H), 8.69-8.73 (m, 1H).

Intermediate C43

Methyl chloro[2-(2,5-difluorophenyl)hydrazono]acetate

Under an argon atmosphere, 3.63 g (24.13 mmol) of methyl 2-chloro-3-oxobutanoate was initially charged to 100 ml of water. 48.90 g (618.19 mmol, 50.00 ml) of pyridine were added at -5°C and stirred at this temperature for 10 minutes. Then, 5.00 g (21.94 mmol) of 2,5-difluorobenzenediazonium tetrafluoroborate was added at -5°C, resulting in the formation of an orange suspension. After stirring at this temperature for 30 minutes, the preparation was diluted with water and extracted three times with dichloromethane. The combined organic phases were washed with saturated sodium chloride solution, dried over sodium sulfate, concentrated on a rotary evaporator, and dried under high vacuum. This yielded 5.52 g of the target compound (97% of theory, purity according to LC/MS = 96%).

LC-MS (Method 1): R _t = 1.03 min; MS (ESIpos): m/z = 249 [M+H] ⁺ .

¹ H NMR (400 MHz, DMSO-d ₆ ): δ [ppm] = 3.85 (s, 3H), 6.88-6.94 (m, 1H), 7.16-7.21 (m, 1H), 7.31-7.37 (m, 1H), 10.00 (s, 1H).

Intermediate C44

Methyl 4-benzoyl-1-(2,5-difluorophenyl)-1H-pyrazole-3-carboxylate

3.50 g (13.52 mmol) of methyl chloro[2-(2,5-difluorophenyl)hydrazono]acetate (96% purity according to LC/MS) was dissolved in 9 ml of pure toluene. 2.61 g (14.87 mmol) of (2E)-3-(dimethylamino)-1-phenylprop-2-en-1-one and 3.01 g (29.73 mmol, 4.14 ml) of triethylamine were added and stirred at room temperature for 16 hours. The reaction mixture was concentrated on a rotary evaporator, and the residue was separated by preparative HPLC (eluent: ACN/water containing 0.1% formic acid, gradient). This yielded 1.79 g (39% of theory) of the target compound.

LC-MS (Method 1): R _t = 1.07 min; MS (ESIpos): m/z = 343 [M+H] ⁺ .

¹ H NMR (400 MHz, DMSO-d ₆ ): δ [ppm] = 3.86 (s, 3H), 7.44-7.50 (m, 1H), 7.55-7.72 (m, 4H), 7.81-7.87 (m, 3H), 8.80 (d, 1H).

Intermediate C45

[4-Benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]methanol

3.18 g (8.92 mmol) of methyl 4-benzoyl-1-(2,5-difluorophenyl)-1H-pyrazole-3-carboxylate (purity = 96% according to LC/MS) was initially charged in 50 mL of trifluoroacetic acid. 8.74 g (75.13 mmol, 12 mL) of triethylsilane was added dropwise and stirred at room temperature for 1 hour. The reaction mixture was concentrated on a rotary evaporator and dried under high vacuum. The resulting residue was taken up in 120 mL of pure THF, and 2.89 g (33.63 mmol, 33.63 mL) of borane-tetrahydrofuran complex was added dropwise at 0°C. Stirring continued overnight. Due to low conversion, an additional 12.33 mL (12.33 mmol) of a 1 M lithium borohydride solution in THF was added. The mixture was stirred at room temperature for 1 hour, at 60°C for 30 minutes, and at 80°C for 2 hours. The mixture was carefully quenched with 60 ml of saturated sodium bicarbonate solution at 0°C. The mixture was extracted twice with in each case 100 ml of ethyl acetate, the combined organic phases were dried over sodium sulfate and concentrated on a rotary evaporator, and the residue was dried under high vacuum. This gave 2.67 g (76% of theory, purity = 96%) of the target compound.

LC-MS (Method 3): R _t = 2.79 min; MS (ESIpos): m/z = 329 [M+H] ⁺ .

¹ H NMR (400 MHz, DMSO-d ₆ ): δ [ppm] = 3.91 (s, 2H), 4.45 (d, 2H), 6.51(s, 1H), 7.18-7.23 (m, 2H), 7.27-7.32 (m, 4H), 7.46-7.53 (m, 1H), 7.60-7.65(m, 1H), 7.95(d, 1H).

Intermediate C46

4-Benzyl-1-(2,5-difluorophenyl)-1H-pyrazole-3-carbaldehyde

Dissolve 2.66 g (8.50 mmol) of [4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]methanol (96% purity) in 150 ml of dichloromethane, and add 4.33 g (10.20 mmol) of Dess-Martin periodinane portionwise. Stir the mixture at room temperature for 2 hours, then add 100 ml of semiconcentrated sodium bicarbonate solution and 100 ml of 10% sodium thiosulfate solution and stir for 20 minutes. Separate the organic phase, dry over sodium sulfate, and concentrate under high vacuum. This yields 2.35 g (88% of theory, 95% purity) of the target compound.

LC-MS (Method 7): R _t = 1.49 min; MS (ESIpos): m/z = 299 [M+H] ⁺ .

¹ H NMR (400 MHz, DMSO-d ₆ ): δ [ppm] = 4.12 (s, 2H), 7.17-7.21 (m, 1H), 7.27-7.31 (m, 4H), 7.37-7.42 (m, 1H), 7.57-7.62 (m, 1H), 7.75-7.78 (m, 1H), 8.22 (d, 1H), 10.06 (s, 1H).

Intermediate C47

(1R)-1-[4-Benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropan-1-amine

Dissolve 2.35 g (7.56 mmol) of 4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazole-3-carbaldehyde in 25 ml of pure THF, and add 1.10 g (9.08 mmol) of (R)-(+)-2-methyl-2-propanesulfenamide and 4.73 g (16.64 mmol) of titanium(IV) isopropoxide. The reaction mixture is stirred at room temperature for 16 hours, after which 20 ml of saturated sodium chloride solution and 30 ml of ethyl acetate are added. Approximately 3 g of celite are then added, and the mixture is boiled under reflux for 1 hour. The organic phase is filtered and separated from the filtrate. The aqueous phase is extracted with ethyl acetate, and the combined organic phases are washed with saturated sodium chloride solution, dried over sodium sulfate, concentrated on a rotary evaporator, and dried under high vacuum. The residue is used without further purification.

Under an argon atmosphere, the residue was dissolved in 60 ml of pure THF and cooled to -78°C. 14.5 ml (23.24 mmol) of tert-butyllithium solution in pentane (c = 1.6 mol/l) was added dropwise. Stir at -78°C for 3 hours, then quenched with 5 ml of methanol and 15 ml of saturated ammonium chloride solution. The reaction mixture was allowed to warm to room temperature while stirring (approximately 30 minutes). Extraction was performed with ethyl acetate, and the organic phase was extracted with saturated sodium chloride solution, concentrated on a rotary evaporator, and dried under high vacuum. The residue was used further without further purification.

The residue was placed in 30 ml of THF and 6 ml of methanol, 6 ml (24.00 mmol) of 4N hydrogen chloride solution in dioxane was added, and the mixture was stirred at room temperature for 1 hour. 15 ml of saturated sodium carbonate solution was then added, and the mixture was extracted with ethyl acetate. The organic phase was separated, concentrated on a rotary evaporator, and dried under high vacuum. The residue was separated by preparative HPLC (eluent: ACN/water, gradient). This yielded two fractions of the target compound. The first fraction yielded 1.31 g (72% of theory, LC/MS purity = 97%), and the second fraction yielded 0.37 g (17% of theory, LC/MS purity = 83%) of the product.

LC-MS (Method 1): R _t = 0.88 min; MS (ESIpos): m/z = 356 [M+H] ⁺ .

¹ H NMR (400 MHz, DMSO-d ₆ ): δ [ppm] = 0.91 (s, 9H), 1.71 (s, 2H), 3.59(s, 1H), 3.87 (s, 2H), 7.17-7.32 (m, 6H), 7.45-7.51 (m, 1H), 7.61-7.65 (m,1H), 7.84(sbr,1H).

Intermediate C48

tert-Butyl (2S)-4-({(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}amino)-2-[(tert-butoxycarbonyl)amino]butanoate

1.28 g (3.35 mmol, LC/MS purity 93%) of (1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropan-1-amine was dissolved in 100 ml of pure dichloromethane. 261 mg (4.35 mmol, 250 μl) of acetic acid and 1.14 g (4.34 mmol) of sodium triacetoxyborohydride were added at room temperature. After stirring for 5 minutes, 1.19 g (4.35 mmol) of tert-butyl (2S)-2-[(tert-butoxycarbonyl)amino]-4-oxobutanoate was added. The mixture was stirred at room temperature for 15 minutes, concentrated on a rotary evaporator, taken up in acetonitrile and water, and purified by preparative HPLC (eluent: ACN/water + 0.1% TFA, gradient). This yielded 1.64 g (80% of theory) of the target compound.

LC-MS (Method 1): R _t = 1.10 min; MS (ESIpos): m/z = 613 [M+H] ⁺ .

¹ H NMR (400 MHz, DMSO-d ₆ ): δ [ppm] = 1.01 (s, 9H), 1.32 (s, 9H), 1.35 (s, 9H), 1.80-1.89 (m, 1H), 2.01-2.11 (m, 1H), 2.54-2.71 (m, 2H), 3.75-3.81(m, 1H), 3.90 (s, 2H), 4.18 (d, 1H), 7.13 (d, 1H), 7.20-7.24 (m, 1H), 7.28-7.34 (m, 5H), 7.52-7.58 (m, 1H), 7.76-7.80 (m, 1H), 8.10 (s br, 1H), 8.23 (sbr, 1H).

Intermediate C49

(2S)-4-[{(1R)-1-[4-Benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-2-[(tert-butoxycarbonyl)amino]butanoic acid

Dissolve 225 mg (0.37 mmol) of tert-butyl (2S)-4-({(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}amino)-2-[(tert-butoxycarbonyl)amino]butanoate in 10 ml of pure dichloromethane, and add 156 mg (1.54 mmol) of triethylamine. Add 125 mg (0.92 mmol) of acetoxyacetyl chloride at 0°C, and stir at room temperature for 16 hours. Add another 251 mg (1.84 mmol) of acetoxyacetyl chloride and 186 mg (1.84 mmol) of triethylamine, and stir at room temperature for 3 hours. Add a small amount of dichloromethane, and wash with saturated sodium bicarbonate solution and saturated sodium chloride solution. Dry the organic phase over sodium sulfate, concentrate on a rotary evaporator, and dry under high vacuum. The residue was taken up in 10 ml of ethanol, 0.91 ml (12.67 mmol) of 40% aqueous methylamine solution was added, and the mixture was stirred at 50°C for 3 hours. The mixture was concentrated on a rotary evaporator, the residue was taken up in dichloromethane, and the organic phase was washed twice with water. The organic phase was dried over sodium sulfate, concentrated on a rotary evaporator, and dried under high vacuum. The residue was taken up in 2 ml of dichloromethane, 2 ml (25.96 mmol) of trifluoroacetic acid was added, and the mixture was stirred at 50°C for 4 hours. The mixture was concentrated on a rotary evaporator, and the residue was dried under high vacuum. The residue was taken up in 10 ml of pure dichloromethane, 298 mg (2.95 mmol) of triethylamine and 429 mg (1.97 mmol) of di-tert-butyl dicarbonate were added, and the mixture was stirred at room temperature for 1 hour. The mixture was concentrated on a rotary evaporator, and the residue was purified by preparative HPLC (eluent: ACN/water, gradient). This yielded 62 mg (27% of theory) of the target compound.

LC-MS (Method 1): R _t = 1.32 min; MS (ESIpos): m/z = 615 [M+H] ⁺ .

¹ H NMR (400 MHz, DMSO-d ₆ ): δ [ppm] = 0.91 (s, 9H), 1.32 (s, 9H), 2.64-2.72 (m, 4H), 3.50-3.58 (m, 1H), 3.72 (dd, 2H), 4.07-4.22 (m, 2H), 4.47-4.54(m, 1H), 5.75 (s, 1H), 6.84-6.89 (m, 1H), 7.15-7.30 (m, 6H), 7.47-7.53 (m,1H), 7.70-7.75 (m, 1H), 8.09-8.13 (m, 1H), 11.66 (s br, 1H).

Intermediate C50

tert-Butyl [(2S)-4-[{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-1-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino}-1-oxobutan-2-yl]carbamate

Dissolve 60 mg (0.1 mmol) of (2S)-4-[{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-2-[(tert-butoxycarbonyl)amino]butanoic acid in 10 mL of pure DMF, and add 74 mg (0.20 mmol) of HATU. Separately, dissolve 74 mg (0.29 mmol) of trifluoroacetic acid/1-(2-aminoethyl)-1H-pyrrole-2,5-dione (1:1) in 2 mL of pure DMF, and add 38 mg (0.29 mmol) of N , N -diisopropylethylamine dropwise to the reaction mixture. Stir at room temperature for 3 days. The mixture is then directly purified by preparative HPLC (eluent: ACN/water + 0.1% TFA, gradient). This gave 9.3 mg (13% of theory) of the target compound.

LC-MS (Method 1): R _t = 1.34 min; MS (ESIpos): m/z = 737 [M+H] ⁺ .

Intermediate C51

N-{(2S)-4-[{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-2-[(tert-butoxycarbonyl)amino]butyryl}-β-alanine

First, intermediate C47 was reductively alkylated with N-{(2S)-2-[(tert-butoxycarbonyl)amino]-4-oxobutanoyl}-β-alanine benzyl ester in a manner analogous to intermediate C2. The secondary amino group was then acylated with 2-chloro-2-oxoethyl acetate as described for intermediate C27, and the two ester groups were then hydrolyzed with 2M lithium hydroxide solution in methanol. This yielded 23 mg of the title compound.

LC-MS (Method 1): R _t = 1.24 min; MS (ESIpos): m/z = 686 (M+H) ⁺ .

Intermediate C52

(1R)-1-[1-Benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropan-1-amine

Initially, 10.00 g (49.01 mmol) of methyl 4-bromo-1H-pyrrole-2-carboxylate was charged into 100.0 ml of DMF, and 20.76 g (63.72 mmol) of cesium carbonate and 9.22 g (53.91 mmol) of benzyl bromide were added. The reaction mixture was stirred at room temperature overnight. The reaction mixture was partitioned between water and ethyl acetate, and the aqueous phase was extracted with ethyl acetate. The combined organic phases were dried over magnesium sulfate, and the solvent was evaporated under vacuum. The formulation was repeated using 90.0 g of methyl 4-bromo-1H-pyrrole-2-carboxylate.

The two combined preparations were purified by preparative RP-HPLC (column: Daiso 300x100; 10µ, flow rate: 250 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 125.15 g (87% of theory) of methyl 1-benzyl-4-bromo-1H-pyrrole-2-carboxylate.

LC-MS (Method 1): R _t = 1.18 min; MS (ESIpos): m/z = 295 [M+H] ⁺ .

Under argon, 4.80 g (16.32 mmol) of methyl 1-benzyl-4-bromo-1H-pyrrole-2-carboxylate was initially charged in DMF, and 3.61 g (22.85 mmol) of (2,5-difluorophenyl)boronic acid, 19.20 ml of saturated sodium carbonate solution, and 1.33 g (1.63 mmol) of [1,1'-bis(diphenylphosphino)ferrocene]-dichloropalladium(II):dichloromethane were added. The reaction mixture was stirred at 85°C overnight. The reaction mixture was filtered through Celite, and the filter cake was washed with ethyl acetate. The organic phase was extracted with water and then washed with saturated NaCl solution. The organic phase was dried over magnesium sulfate, and the solvent was evaporated under vacuum. The residue was purified on silica gel (eluent: cyclohexane/ethyl acetate 100:3). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 3.60 g (67% of theory) of the compound methyl 1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrole-2-carboxylate.

LC-MS (Method 7): R _t = 1.59 min; MS (ESIpos): m/z = 328 [M+H] ⁺ .

Initially, 3.60 g (11.00 mmol) of methyl 1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrole-2-carboxylate was charged in 90.0 ml of THF, and 1.04 g (27.50 mmol) of lithium aluminum hydride (2.4 M in THF) was added at 0°C. The reaction mixture was stirred at 0°C for 30 minutes. Saturated potassium sodium tartrate solution was added at 0°C, and ethyl acetate was added to the reaction mixture. The organic phase was extracted three times with saturated potassium sodium tartrate solution. The organic phase was washed once with saturated NaCl solution and dried over magnesium sulfate. The solvent was evaporated under vacuum, and the residue was dissolved in 30.0 ml of dichloromethane. 3.38 g (32.99 mmol) of manganese(IV) oxide was added, and the mixture was stirred at room temperature for 48 hours. An additional 2.20 g (21.47 mmol) of manganese(IV) oxide was added, and the mixture was stirred at room temperature overnight. The reaction mixture was filtered through celite, and the filter cake was washed with dichloromethane. The solvent was evaporated in vacuo and the residue, 2.80 g (1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrole-2-carbaldehyde), was used in the next step of the synthesis without further purification.

LC-MS (Method 7): R _t = 1.48 min; MS (ESIpos): m/z = 298 [M+H] ⁺ .

28.21 g (94.88 mmol) of 1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrole-2-carbaldehyde and 23.00 g (189.77 mmol) of (R)-2-methylpropane-2-sulfenamide were initially charged in 403.0 ml of pure THF. 67.42 g (237.21 mmol) of titanium(IV) isopropoxide were added and stirred at room temperature overnight. 500.0 ml of saturated NaCl solution and 1000.0 ml of ethyl acetate were added and stirred at room temperature for 1 hour. The mixture was filtered through Celite, and the filtrate was washed twice with saturated NaCl solution. The organic phase was dried over magnesium sulfate, and the solvent was evaporated under vacuum. The residue was purified using a Biotage Isolera (silica gel, column 1500 + 340 g SNAP, flow rate 200 ml/min, ethyl acetate/cyclohexane 1:10).

LC-MS (Method 7): R _t = 1.63 min; MS (ESIpos): m/z = 401 [M+H] ⁺ .

25.00 g (62.42 mmol) of (R)-N-{(E/Z)-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]methylene}-2-methylpropane-2-sulfenamide were initially placed in pure THF under argon and cooled to -78°C. 12.00 g (187.27 mmol) of tert-butyllithium (1.7 M solution in pentane) were then added at -78°C and stirred at this temperature for 3 hours. 71.4 ml of methanol and 214.3 ml of saturated ammonium chloride solution were then added successively at -78°C, and the reaction mixture was allowed to warm to room temperature and stirred at room temperature for 1 hour. The mixture was diluted with ethyl acetate and washed with water. The organic phase was dried over magnesium sulfate, and the solvent was evaporated under vacuum. The residue (R)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2-methylpropane-2-sulfenamide was used in the next step of the synthesis without further purification.

LC-MS (Method 6): R _t = 2.97 min; MS (ESIpos): m/z = 459 [M+H] ⁺ .

28.00 g (61.05 mmol) of (R)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2-methylpropane-2-sulfenamide were initially charged in 186.7 ml of 1,4-dioxane, followed by the addition of 45.8 ml of a 4.0 M HCl/1,4-dioxane solution. The reaction mixture was stirred at room temperature for 2 hours, and the solvent was evaporated under vacuum. The residue was purified by preparative RP-HPLC (column: Kinetix 100x30; flow rate: 60 ml/min, MeCN/water). Acetonitrile was evaporated under vacuum, and dichloromethane was added to the aqueous residue. The organic phase was washed with sodium bicarbonate solution and dried over magnesium sulfate. The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 16.2 g (75% of theory) of the title compound.

LC-MS (Method 6): R _t = 2.10 min; MS (ESIpos): m/z = 338 [M-NH ₂ ] ⁺ , 709 [2M+H] ⁺ .

¹ H NMR (400 MHz, DMSO-d ₆ ): δ [ppm] = 0.87 (s, 9H), 1.53 (s, 2H), 3.59(s, 1H), 5.24 (d, 2H), 6.56 (s, 1H), 6.94 (m, 1H), 7.10 (d, 2H), 7.20 (m,1H), 7.26 (m, 2H), 7.34 (m, 2H), 7.46 (m, 1H).

Intermediate C53

(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}butanoic acid

First, intermediate C52 is similar to C2 with (2S)-2-{[(benzyloxy)carbonyl]amino}-4-oxobutyric acid benzyl ester reductive alkylation.This secondary amino group is subsequently as described in intermediate C27 with acetic acid 2-chloro-2-oxoethyl ester acylation, and these two ester groups are then hydrolyzed with 2M lithium hydroxide solution in methanol.The intermediate thus obtained is dissolved in ethanol, adds palladium carbon (10%) and hydrogenates 1 hour with hydrogen at room temperature under standard pressure.The deprotected compound is placed in dioxane/water 2:1 and in the final step, in the presence of N , N -diisopropylethylamine, uses chlorocarbonic acid 9H-fluorene-9-ylmethyl ester to introduce Fmoc protecting group.

LC-MS (Method 1): R _t = 1.37 min; MS (ESIpos): m/z = 734 (MH) ^- .

Intermediate C54

N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}butyryl]-β-alanine

First, intermediate C52 is similar to intermediate C2 with N-[(2S)-2-{[(benzyloxy)carbonyl]amino}-4-oxobutanoyl]-β-alanine benzyl ester reductive alkylation.The secondary amino group is subsequently acylated with 2-chloro-2-oxoethyl acetate as described for intermediate C27.The intermediate thus obtained is dissolved in ethanol, palladium carbon (10%) is added and hydrogenated with hydrogen at room temperature under standard pressure for 1 hour.The two ester groups are then hydrolyzed with 2M lithium hydroxide solution in methanol.The deprotected compound is placed in dioxane/water 2:1 and in the final step, 9H-fluorene-9-ylmethyl chlorocarbonate is used to introduce the Fmoc protecting group in the presence of N , N -diisopropylethylamine.This produces 48 milligrams of title compound.

LC-MS (Method 1): R _t = 1.38 min; MS (ESIpos): m/z = 807 (M+H) ⁺ .

Intermediate C55

2-[3-({(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}amino)propyl]-1H-isoindole-1,3(2H)-dione

340 mg (0.96 mmol) of (1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropan-1-amine was dissolved in 7 ml of pure DCM, and 69 mg (1.15 mmol, 60 μL) of acetic acid and 284 mg (1.34 mmol) of sodium triacetoxyborohydride were added at room temperature. The mixture was stirred for 15 minutes, and then 233 mg (1.15 mmol) of 3-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)propanal was added. The mixture was stirred at room temperature for 4.5 hours. An additional 233 mg (1.15 mmol) of 3-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)propanal, 69 mg (1.15 mmol, 60 μl) of acetic acid, and 284 mg (1.34 mmol) of sodium triacetoxyborohydride were added, and the mixture was stirred at room temperature for 7 hours. Ethyl acetate was added to the reaction mixture, which was then washed with saturated sodium carbonate solution. The organic phase was concentrated, and the residue was purified twice by preparative HPLC [1.) eluent: ACN/water + 0.1% TFA, gradient; 2.) eluent: ACN/water + 1% TFA + 1.0% NEt ₃ ]. This yielded 108 mg (21% of theory) of the target compound.

LC-MS (method 1): R _t = 0.96 min; MS (ESIpos): m/z = 543 [M+H] ⁺ .

Intermediate C56

2-({(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}[3-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)propyl]amino)-2-oxoethyl acetate

Initially, 102 mg (0.19 mmol) of 2-[3-({(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}amino)propyl]-1H-isoindole-1,3(2H)-dione was charged in 2 ml of pure DCM, and 44 mg (0.43 mmol) of triethylamine was added at room temperature. At 0°C, 31 mg (0.23 mmol) of 2-chloro-2-oxoethyl acetate dissolved in 1 ml of pure DCM was added. The mixture was stirred at room temperature for 40 minutes. Another 26 mg of 2-chloro-2-oxoethyl acetate was dissolved in 0.5 ml of pure DCM, 19 mg (0.19 mmol) of triethylamine was added, and the mixture was stirred at room temperature for 60 minutes.

Water was added, the mixture was concentrated on a rotary evaporator, and the residue was purified by preparative HPLC (eluent: ACN/water + 0.1% TFA, gradient). This gave 106 mg (88% of theory) of the target compound.

LC-MS (Method 1): R _t = 1.37 min; MS (ESIpos): m/z = 643 [M+H] ⁺ .

Intermediate C57

Trifluoroacetic acid/tert-butyl {(2S)-1-[(2-aminoethyl)amino]-4-[{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-1-oxobutan-2-yl}carbamate (1:1)

The title compound was prepared by coupling intermediate C49 with 9H-fluoren-9-ylmethyl (2-aminoethyl)carbamate in the presence of HATU and subsequent cleavage of the Fmoc protecting group with piperidine according to standard procedures. This yielded 14 mg of the title compound (40% of theory over 2 steps).

LC-MS (Method 1): R _t = 0.98 min; MS (ESIpos): m/z = 657 (M+H) ⁺ .

Intermediate C58

(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoic acid

First, intermediate C52 was reductively alkylated with (2S)-2-{[(benzyloxy)carbonyl]amino}-4-oxobutanoic acid benzyl ester in a manner analogous to intermediate C2. The secondary amino group was then acylated with 2-chloro-2-oxoethyl acetate as described for intermediate C27, and the two ester groups were then hydrolyzed with 2M lithium hydroxide solution in methanol. The intermediate thus obtained was dissolved in ethanol, palladium on carbon (10%) was added, and hydrogenated with hydrogen at room temperature under standard pressure for 1 hour.

500 mg (0.886 mmol) of this fully deprotected intermediate were placed in 60 ml of dioxane, and 253 mg (0.975 mmol) of 1-({[2-(trimethylsilyl)ethoxy]carbonyl}oxy)pyrrolidine-2,5-dione and 198 μl of triethylamine were added. After stirring at room temperature for 24 hours, the preparation was concentrated, and the residue was purified by preparative HPLC. Combining the appropriate fractions, concentrating under vacuum, and drying under high vacuum yielded 312 mg (50% of theory) of the title compound.

LC-MS (Method 5): R _t = 4.61 min; MS (ESIpos): m/z = 658 (M+H) ^- .

Intermediate C59

(2S)-4-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}[(2S)-2-methoxypropionyl]amino)-2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}butanoic acid

First, the secondary amino group of benzyl (2S)-4-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)-2-{[(benzyloxy)carbonyl]amino}butanoate was acylated with (2S)-2-methoxypropionyl chloride (an intermediate of intermediate C53) in the presence of triethylamine as described for intermediate C53. The resulting intermediate was taken up in ethanol, palladium on carbon (10%) was added, and hydrogenated with hydrogen at standard pressure for 1 hour at room temperature. The deprotected compound was placed in dioxane/water 2:1 and, in a final step, the Fmoc protecting group was introduced using 9H-fluoren-9-ylmethyl chlorocarbonate in the presence of N , N -diisopropylethylamine.

LC-MS (Method 1): R _t = 1.39 min; MS (ESIpos): m/z = 764 (MH) ^- .

Intermediate C60

(2S)-4-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}[(2S)-2-methoxypropionyl]amino)-2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}butanoic acid

The synthesis was carried out similarly to intermediate C53.

LC-MS (Method 1): R _t = 1.41 min; MS (ESIpos): m/z = 750 (M+H) ⁺ .

Intermediate C61

N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoyl]-β-alanine

The title compound was prepared by coupling 60 mg (0.091 mmol) of intermediate C58 with β-alanine methyl ester followed by ester cleavage using 2M lithium hydroxide solution. This yielded 67 mg (61% of theory) of the title compound over 2 steps.

LC-MS (Method 1): R _t = 1.29 min; MS (ESIpos): m/z = 729 (M+H) ⁺ .

Intermediate C62

N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoyl]-D-alanine

The title compound was prepared analogously to intermediate C61 from intermediate C58 and D-alanine methyl ester.

LC-MS (Method 1): R _t = 1.32 min; MS (ESIpos): m/z = 729 (M+H) ⁺ .

Intermediate C63

Trifluoroacetic acid/tert-butyl {(2S)-1-[(2-aminoethyl)amino]-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-1-oxobutan-2-yl}carbamate (1:1)

The synthesis of this intermediate began in the first step by coupling 50 mg (0.075 mmol) of intermediate C3 with 26.2 mg (0.082 mmol) of 9H-fluoren-9-ylmethyl (2-aminoethyl)carbamate hydrochloride (1:1) in the presence of 28.7 mg (0.15 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 22.9 mg (0.15 mmol) of 1-hydroxy- 1H -benzotriazole hydrate, and 39 μl of N , N -diisopropylethylamine. After stirring at room temperature for 18 hours, the mixture was concentrated and the residue was purified by preparative HPLC. This yielded 45 mg (65% of theory) of this intermediate.

LC-MS (Method 1): R _t = 1.51 min; MS (ESIpos): m/z = 921 (M+H) ⁺ .

45 mg (0.049 mmol) of this intermediate was dissolved in 10 ml of ethanol, and 176 μl of a 40% aqueous methylamine solution was added. The mixture was stirred at 50°C, and the same amount of methylamine solution was added after 6 and 9 hours. After stirring at 50°C for a further 14 hours, an additional 700 μl of methylamine solution was added, and after stirring for a further 24 hours, the mixture was concentrated. The residue was taken up in DCM and washed with water. The organic phase was concentrated, and the residue was purified by preparative HPLC. Concentration of the appropriate fractions and drying of the residue under high vacuum yielded 32 mg (99% of theory) of tert-butyl {(2S)-1-[(2-aminoethyl)amino]-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-1-oxobutan-2-yl}carbamate.

LC-MS (Method 1): R _t = 0.95 min; MS (ESIpos): m/z = 657 (M+H) ⁺ .

Intermediate C64

Trifluoroacetic acid/2-(trimethylsilyl)ethyl {(2S)-1-[(2-aminoethyl)amino]-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-1-oxobutan-2-yl}carbamate (1:1)

The title compound was prepared from intermediate C58 in analogy to intermediate C63.

HPLC (Method 11): R _t = 2.4 min;

LC-MS (Method 1): R _t = 1.01 min; MS (ESIpos): m/z = 700 (M+H) ⁺ .

Intermediate C65

(8S)-8-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-(ethanolyl)amino]ethyl}-2,2-dimethyl-6,11-dioxo-5-oxa-7,10-diaza-2-sila-tetradec-14-oic acid

Initially, 215 mg (0.59 mmol) of intermediate L66 was charged to 25 ml of dichloromethane, and 377 mg (0.89 mmol) of Dess _- Martin periodinane and 144 μl (1.78 mmol) of pyridine were added. Stirring was carried out at room temperature for 30 minutes. The preparation was then diluted with 300 ml of dichloromethane, and the organic phase was washed twice each with 10% _{Na₂S₂O₃} solution, 10% citric _acid solution, and saturated sodium bicarbonate solution. The organic phase was dried over magnesium sulfate, and the solvent was evaporated under vacuum. This yielded 305 mg of aldehyde, which was reacted without further purification.

175 mg (0.49 mmol) of Intermediate C52 was dissolved in 50 ml of dichloromethane, and 147 mg (0.69 mmol) of sodium triacetoxyborohydride and 32.5 μL of acetic acid were added. After stirring at room temperature for 5 minutes, 214 mg (0.593 mmol) of the above-mentioned aldehyde was added, and the mixture was stirred at room temperature overnight. Instead of the expected product, 2-(trimethylsilyl)ethyl [(2S)-4-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)-1-(2,5-dioxopyrrolidin-1-yl)butan-2-yl]carbamate was formed. Since this imide can also be converted into the title compound, the mixture was concentrated, and the residue was purified by preparative HPLC. After combining the appropriate imide-containing fractions, the solvent was evaporated under vacuum and the residue was dried under high vacuum. This yielded 195 mg (58%) of the above-specified imide.

LC-MS (Method 5): R _t = 3.32 min; MS (ESIpos): m/z = 667 (M+H) ⁺ .

65 mg (97.5 μmol) of this imide was dissolved in 15 ml of dichloromethane, and 367 μl (3.4 mmol) of acetoxyacetyl chloride and 595 μl of N , N -diisopropylethylamine were added. After stirring at room temperature for 30 minutes, the mixture was concentrated under vacuum without heating, and the residue was purified by preparative HPLC. Appropriate fractions were combined, evaporated, and dried under high vacuum to yield 28 mg (37% of theory) of (8S)-11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-8-[(2,5-dioxopyrrolidin-1-yl)methyl]-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl acetate.

LC-MS (Method 1): R _t = 1.44 min; MS (ESIpos): m/z = 767 (M+H) ⁺ .

28 mg (37 μmol) of this intermediate were dissolved in 3 ml of methanol, and 548 μl of 2M lithium hydroxide solution were added. After stirring at room temperature for 10 minutes, the preparation was adjusted to pH 4 with trifluoroacetic acid and then concentrated. The residue was purified by preparative HPLC. Appropriate fractions were combined, the solvent was evaporated, and the residue was dried under high vacuum to yield 26 mg (96% of theory) of the title compound as a white solid.

LC-MS (Method 1): R _t = 1.33 min; MS (ESIpos): m/z = 743 (M+H) ⁺ .

Intermediate C66

2-(Trimethylsilyl)ethyl [(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-1-{[2-(glycylamino)ethyl]amino}-1-oxobutan-2-yl]carbamate

First, trifluoroacetic acid/benzyl {2-[(2-aminoethyl)amino]-2-oxoethyl}carbamate (1:1) was prepared from N-[(benzyloxy)carbonyl]glycine and tert-butyl (2-aminoethyl)carbamate according to the classical method of peptide chemistry (HATU coupling and Boc cleavage).

13 mg (0.036 mmol) of this intermediate and 25 mg (0.033 mmol) of intermediate C58 were placed in 3 ml of DMF, and 19 mg (0.05 mmol) of HATU and 17 μl of N , N -diisopropylethylamine were added.

After stirring at room temperature for 10 minutes, the mixture was concentrated and the residue was purified by preparative HPLC. This gave 17.8 mg (60% of theory) of the intermediate.

LC-MS (Method 1): R _t = 1.36 min; MS (ESIpos): m/z = 891 (M+H) ⁺ .

17 mg (0.019 mmol) of this intermediate was dissolved in 10 ml of ethanol, palladium on carbon (10%) was added, and the mixture was hydrogenated with hydrogen at standard pressure at room temperature for 2 hours. The catalyst was filtered off, the solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 9 mg (62% of theory) of the title compound.

LC-MS (Method 1): R _t = 1.03 min; MS (ESIpos): m/z = 757 (M+H) ⁺ .

Intermediate C67

9H-Fluoren-9-ylmethyl [3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)propyl]carbamate

Initially, 605.3 mg (1.71 mmol) of (1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropan-1-amine (Intermediate C52) was charged in 10.0 mL of dichloromethane. 506.7 mg (2.39 mmol) of sodium triacetoxyborohydride and 117.9 mg (1.96 mmol) of acetic acid were added and stirred at room temperature for 5 minutes. 580.0 mg (1.96 mmol) of 9H-fluoren-9-ylmethyl (3-oxopropyl)carbamate (Intermediate L70) was dissolved in 10.0 mL of dichloromethane, and the reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with ethyl acetate, and the organic phase was washed twice with saturated sodium carbonate solution and twice with saturated NaCl solution. The organic phase was dried over magnesium sulfate, and the solvent was evaporated under vacuum. The residue was purified on silica gel (eluent: cyclohexane/ethyl acetate 3:1). The solvent was evaporated under vacuum and the residue was dried under high vacuum. This gave 514.7 mg (46% of theory) of the title compound.

LC-MS (Method 1): R _t = 1.10 min; MS (ESIpos): m/z = 634 (M+H) ⁺ .

Intermediate C68

tert-Butyl [3-({(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}amino)propyl]carbamate

The synthesis was performed similarly to the synthesis of compound intermediate C67.

1000.0 mg (2.81 mmol) (1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropan-1-amine (Intermediate C47)

835.0 mg (3.94 mmol) sodium triacetoxyborohydride

194.0 mg (3.24 mmol) acetic acid

560.0 mg (3.24 mmol) tert-butyl (3-oxopropyl)carbamate

This gave 695.8 mg (48% of theory) of the title compound.

LC-MS (Method 1): R _t = 1.02 min; MS (ESIpos): m/z = 513 (M+H) ⁺ .

Intermediate C69

11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-14-thia-7,11-diaza-2-silaheptadecane-17-oic acid

117.0 mg (0.19 mmol) of 2-(trimethylsilyl)ethyl {3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(chloroacetyl)amino]propyl}carbamate (Intermediate C70) and 21.6 mg (0.20 mmol) of 3-sulfanylpropionic acid were initially charged in 3.0 ml of methanol, 89.5 mg (0.65 mmol) of potassium carbonate were added and stirred at 50° C. for 4 hours. The reaction mixture was diluted with ethyl acetate, and the organic phase was washed with water and saturated NaCl solution. The organic phase was dried over magnesium sulfate, the solvent was evaporated under vacuum, and the residue was dried under high vacuum. The residue was used in the next step of the synthesis without further purification. This gave 106.1 mg (73% of theory) of the title compound.

LC-MS (Method 1): R _t = 1.42 min; MS (ESIneg): m/z = 700 (MH) ^- .

Intermediate C70

2-(Trimethylsilyl)ethyl 3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(chloroacetyl)amino]propyl}carbamate

908.1 mg (1.63 mmol) of 2-(trimethylsilyl)ethyl 3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)propyl]carbamate (see Synthesis of Intermediate C11) and 545.6 mg (5.39 mmol) of triethylamine were initially charged in 10.0 ml of dichloromethane and cooled to 0°C. At this temperature, 590.5 mg (5.23 mmol) of chloroacetyl chloride were added and stirred at room temperature overnight. The reaction mixture was diluted with ethyl acetate, and the organic phase was washed three times with saturated sodium bicarbonate solution and saturated ammonium chloride solution. The organic phase was washed with saturated NaCl solution and dried over magnesium sulfate. The residue was purified by preparative RP-HPLC (column: Reprosil 250x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 673.8 mg (65% of theory) of the title compound.

LC-MS (Method 1): R _t = 1.53 min; MS (ESIneg): m/z = 676 (M+HCOO ^- ) ^- .

Intermediate C71

S-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl)-L-cysteine/trifluoroacetic acid (1:1)

536.6 mg (4.43 mmol) of L-cysteine and 531.5 mg (6.33 mmol) of sodium bicarbonate were suspended in 2.5 ml of water. 400.0 mg (0.63 mmol) of 2-(trimethylsilyl)ethyl 3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(chloroacetyl)amino]propyl}carbamate (Intermediate C70) and 1.16 g (7.59 mmol) of 1,8-diazabicyclo[5.4.0]undec-7-ene, dissolved in 25.0 ml of isopropanol, were added. The reaction mixture was stirred at 50°C for 1.5 hours. Ethyl acetate was added to the reaction mixture, and the organic phase was washed repeatedly with saturated sodium bicarbonate solution and once with saturated NaCl solution. The organic phase was dried over magnesium sulfate, the solvent was evaporated under vacuum, and the residue was dried under high vacuum. The residue was purified by preparative RP-HPLC (column: Reprosil 250x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 449.5 mg (86% of theory) of the title compound.

LC-MS (Method 1): R _t = 1.20 min; MS (ESIpos): m/z = 717 (M+H) ⁺ .

Intermediate C72

(9S)-9-{[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]methyl}-2,2-dimethyl-6,11-dioxo-5-oxa-7,10-diaza-2-silanetetaracan-14-oic acid

Initially, 90 mg (0.212 mmol) of intermediate L72 was dissolved in 6 ml of dichloromethane, and 86 μl (1.06 mmol) of pyridine and 135 mg (0.318 mmol) of Dess _- Martin periodinane were added. The mixture was stirred at room temperature for 30 minutes. The mixture was then diluted with 30 ml _of dichloromethane, and the organic phase was washed twice with 10% _{Na₂S₂O₃} solution and once with 5% citric acid solution. The organic phase was dried over magnesium sulfate, and the solvent was evaporated under vacuum. The aldehyde thus obtained was reacted without further purification.

63 mg (0.177 mmol) of Intermediate C52 was dissolved in 15 ml of dichloromethane, and 52.4 mg (0.247 mmol) of sodium triacetoxyborohydride and 20.2 μL of acetic acid were added. After stirring at room temperature for 5 minutes, 89.6 mg (0.212 mmol) of the above-mentioned aldehyde was added, and the mixture was stirred at room temperature for 20 minutes. The mixture was concentrated under vacuum, and the residue was purified by preparative HPLC. After combining the appropriate fractions, the solvent was evaporated under vacuum, and the residue was lyophilized from acetonitrile/water. This gave 71 mg (53% of theory over 2 steps) of benzyl (9R)-9-[({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)methyl]-2,2-dimethyl-6,11-dioxo-5-oxa-7,10-diaza-2-silanetratetradec-14-oate.

LC-MS (Method 1): R _t = 1.21 min; MS (ESIpos): m/z = 761 (M+H) ⁺ .

70 mg (92 μmol) of this intermediate was dissolved in 15 ml of dichloromethane, cooled to 10°C, and 54 μl of triethylamine and 25.5 μl (0.23 mmol) of acetoxyacetyl chloride were added. After stirring at room temperature for 1 hour, the same amount of acid chloride and triethylamine was added, and after stirring at room temperature for another hour, more was added. The preparation was then stirred at room temperature for another 30 minutes before being concentrated under vacuum, and the residue was purified by preparative HPLC. The appropriate fractions were combined, and after evaporation of the solvent and lyophilization of the residue from acetonitrile/water, 46.5 mg (59% of theory) of the acylated intermediate was obtained.

LC-MS (Method 1): R _t = 1.53 min; MS (ESIpos): m/z = 861 (M+H) ⁺ .

46 mg (53 μmol) of this intermediate were dissolved in 5 ml of methanol and 2.7 ml of 2M lithium hydroxide solution were added. After stirring at room temperature for 10 minutes, the preparation was adjusted to pH 3-4 with acetic acid and then diluted with 15 ml of water. The aqueous phase was extracted with ethyl acetate, and the organic phase was dried over magnesium sulfate and concentrated. The residue was lyophilized from acetonitrile/water and dried under high vacuum to yield 37 mg (90% of theory) of the title compound as a white solid.

LC-MS (Method 1): R _t = 1.32 min; MS (ESIpos): m/z = 729 (M+H) ⁺ .

Intermediate C73

S-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl)-N-[3-(trimethylsilyl)propionyl]-L-cysteine

619 mg (0.86 mmol) of S-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl)-L-cysteine/trifluoroacetic acid (1:1) (Intermediate C71) were initially charged in 8.8 ml of dichloromethane, and 87 mg (0.86 mmol) of triethylamine and 224 mg (0.86 mmol) of N-[2-(trimethylsilyl)ethoxycarbonyloxy]pyrrolidine-2,5-dione were added. After 1 hour, 45 mg (0.17 mmol) of N-[2-(trimethylsilyl)ethoxycarbonyloxy]pyrrolidine-2,5-dione were added. The reaction mixture was stirred at room temperature for 1 hour. The mixture was concentrated under vacuum, and the residue was taken up in dichloromethane. The organic phase was then washed twice with water and saturated sodium bicarbonate solution. The organic phase was dried over magnesium sulfate, concentrated on a rotary evaporator, and dried under high vacuum. The residue was used further without further purification. This yielded 602 mg (71%, 87% purity) of the title compound.

LC-MS (Method 1): R _t = 1.58 min; MS (ESIpos): m/z = 861 (M+H) ⁺ .

Intermediate L1

Trifluoroacetic acid/N-(2-aminoethyl)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamide (1:1)

The title compound was prepared by classical methods of peptide chemistry from commercially available (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetic acid and tert-butyl (2-aminoethyl)carbamate.

HPLC (Method 11): R _t = 0.19 min;

LC-MS (Method 1): R _t = 0.17 min; MS (ESIpos): m/z = 198 (M+H) ⁺ .

Intermediate L2

Trifluoroacetic acid/rel-(1R,2S)-2-amino-N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]cyclopentanecarboxamide (1:1)

The title compound was prepared from 50 mg (0.214 mmol) of commercially available cis-2-[(tert-butoxycarbonyl)amino]-1-cyclopentanecarboxylic acid and 60 mg (0.235 mmol) of the likewise commercially available trifluoroacetic acid/1-(2-aminoethyl)-1H-pyrrole-2,5-dione (1:1) by coupling with EDC/HOBT and subsequent deprotection with TFA. This gave 36 mg (38% of theory over 2 steps) of the title compound.

HPLC (Method 11): R _t = 0.2 min;

LC-MS (Method 1): R _t = 0.17 min; MS (ESIpos): m/z = 252 (M+H) ⁺ .

Intermediate L3

Trifluoroacetic acid/(1S,2R)-2-amino-N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]cyclopentanecarboxamide (1:1)

The title compound was prepared from 50 mg (0.214 mmol) of commercially available (1S,2R)-2-[(tert-butoxycarbonyl)amino]cyclopentanecarboxylic acid and 72 mg (0.283 mmol) of the likewise commercially available trifluoroacetic acid/1-(2-aminoethyl)-1H-pyrrole-2,5-dione (1:1) by coupling with EDC/HOBT and subsequent deprotection with TFA. This gave 13 mg (16% of theory over 2 steps) of the title compound.

HPLC (Method 11): R _t = 0.2 min;

LC-MS (Method 1): R _t = 0.2 min; MS (ESIpos): m/z = 252 (M+H) ⁺ .

Intermediate L4

Trifluoroacetic acid/N-(2-aminoethyl)-4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)cyclohexanecarboxamide (1:1)

The title compound was prepared by classical methods of peptide chemistry from commercially available 1-[(4-{[(2,5-dioxopyrrolidin-1-yl)oxy]carbonyl}cyclohexyl)methyl]-1H-pyrrole-2,5-dione and tert-butyl (2-aminoethyl)carbamate.

HPLC (Method 11): R _t = 0.26 min;

LC-MS (Method 1): R _t = 0.25 min; MS (ESIpos): m/z = 280 (M+H) ⁺ .

Intermediate L5

Trifluoroacetic acid/N-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl]-β-alaninamide (1:1)

The title compound was prepared by classical methods of peptide chemistry from commercially available 1-(4-aminophenyl)-1H-pyrrole-2,5-dione and N-(tert-butoxycarbonyl)-β-alanine.

HPLC (Method 11): R _t = 0.22 min;

LC-MS (Method 1): R _t = 0.22 min; MS (ESIpos): m/z = 260 (M+H) ⁺ .

Intermediate L6

Trifluoroacetic acid/N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-L-alanyl-L-lysine tert-butyl ester (1:1)

The title compound was prepared by initially coupling commercially available 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid with the partially protected peptide L-valyl-L-alanyl-N6-(tert-butoxycarbonyl)-L-lysine tert-butyl ester prepared by classical methods of peptide chemistry in the presence of EDC/HOBT. This was followed by deprotection at the amino group under mild conditions by stirring in 5% trifluoroacetic acid in DCM at room temperature, which gave the title compound in 37% yield.

HPLC (Method 11): R _t = 1.29 min;

LC-MS (Method 1): R _t = 0.62 min; MS (ESIpos): m/z = 566 (M+H) ⁺ .

Intermediate L7

Trifluoroacetic acid/β-alanyl-L-valyl-N ⁵ -carbamoyl-N-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl]-L-ornithinamide (1:1)

The title compound was prepared according to classical methods of peptide chemistry from commercially available 1-(4-aminophenyl)-1H-pyrrole-2,5-dione by sequential coupling with N2-(tert-butoxycarbonyl)-N5-carbamoyl-L-ornithine in the presence of HATU, deprotection with TFA, coupling with N-(tert-butoxycarbonyl)-L-valine 2,5-dioxopyrrolidin-1-yl ester, deprotection with TFA, coupling with N-(tert-butoxycarbonyl)-β-alanine 2,5-dioxopyrrolidin-1-yl ester, and further deprotection with TFA. This yielded 32 mg of the title compound.

HPLC (Method 11): R _t = 0.31 min;

LC-MS (Method 1): R _t = 0.47 min; MS (ESIpos): m/z = 516 (M+H) ⁺ .

Intermediate L8

Trifluoroacetic acid/L-alanyl-N ⁵ -carbamoyl-N-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl]-L-ornithinamide (1:1)

The title compound was prepared according to classical methods of peptide chemistry from commercially available 1-(4-aminophenyl)-1H-pyrrole-2,5-dione by sequential coupling with N ² -(tert-butoxycarbonyl)-N ⁵ -carbamoyl-L-ornithine in the presence of HATU, deprotection with TFA, coupling with N-(tert-butoxycarbonyl)-L-alanine 2,5-dioxopyrrolidin-1-yl ester and further deprotection with TFA. This gave 171 mg of the title compound.

HPLC (Method 11): R _t = 0.23 min;

LC-MS (Method 7): R _t = 0.3 min; MS (ESIpos): m/z = 417 (M+H) ⁺ .

Intermediate L9

Trifluoroacetic acid/β-alanyl-L-valyl-N ⁵ -carbamoyl-N-[4-(2-methoxy-2-oxoethyl)phenyl]-L-ornithinamide (1:1)

The title compound was prepared analogously to intermediate L7 from commercially available methyl (4-aminophenyl)acetate. This gave 320 mg of the title compound.

HPLC (Method 11): R _t = 0.45 min;

LC-MS (Method 1): R _t = 0.48 min; MS (ESIpos): m/z = 493 (M+H) ⁺ .

Intermediate L10

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-L-alanyl-rel-N ⁶ -{[(1R,2S)-2-aminocyclopentyl]carbonyl}-L-lysine/trifluoroacetic acid (1:2)

The title compound was prepared from intermediate L6 by coupling with cis-2-[(tert-butoxycarbonyl)amino]-1-cyclopentanecarboxylic acid using EDC/HOBT and subsequent deprotection with TFA. This gave 12 mg (52% of theory over 2 steps) of the title compound.

HPLC (Method 11): R _t = 1.45 min;

LC-MS (Method 1): R _t = 0.73 min; MS (ESIpos): m/z = 677 (M+H) ⁺ .

Intermediate L11

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-L-alanyl-N ⁶ -{[(1S,2R)-2-aminocyclopentyl]carbonyl}-L-lysine/trifluoroacetic acid (1:2)

The title compound was prepared from intermediate L6 by coupling with (1S,2R)-2-[(tert-butoxycarbonyl)amino]cyclopentanecarboxylic acid using EDC/HOBT and subsequent deprotection with TFA. This gave 11 mg (39% of theory over 2 steps) of the title compound.

HPLC (Method 11): R _t = 1.45 min;

LC-MS (Method 1): R _t = 0.74 min; MS (ESIpos): m/z = 677 (M+H) ⁺ .

Intermediate L12

Trifluoroacetic acid/1-[2-(2-aminoethoxy)ethyl]-1H-pyrrole-2,5-dione (1:1)

381 mg (2.46 mmol) of methyl 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate was added to 228 mg (1.12 mmol) of tert-butyl [2-(2-aminoethoxy)ethyl]carbamate dissolved in 7 ml of dioxane/water (1:1). 1.2 ml of saturated sodium bicarbonate solution was then added, and the preparation was stirred at room temperature. After stirring for a total of 5 days and two additional additions of the same amount of sodium bicarbonate solution, the preparation was worked up by acidification with trifluoroacetic acid, concentration on a rotary evaporator, and purification by preparative HPLC. The appropriate fractions were combined, the solvent removed under vacuum, and the residue lyophilized from acetonitrile/water (1:1).

The residue was taken up in 3 ml of dichloromethane and 1 ml of trifluoroacetic acid was added. After stirring at room temperature for 15 minutes, the solvent was removed under vacuum and the residue was lyophilized from acetonitrile/water 1:1. This gave 70 mg (67% of theory over 2 steps) of the title compound as a resinous residue.

HPLC (Method 11): R _t = 0.2 min;

LC-MS (Method 1): R _t = 0.18 min; MS (ESIpos): m/z = 185 (M+H) ⁺ .

Intermediate L13

Trifluoroacetic acid/N2-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-lysine tert-butyl ester (1:1)

The title compound was prepared by coupling (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetic acid with N6-(tert-butoxycarbonyl)-L-lysine tert-butyl ester hydrochloride (1:1) in the presence of EDC/HOBT and subsequent mild cleavage of the tert-butoxycarbonyl protecting group in analogy to intermediate L6.

HPLC (Method 11): R _t = 0.42 min;

LC-MS (Method 1): R _t = 0.43 min; MS (ESIpos): m/z = 340 (M+H) ⁺ .

Intermediate L14

Trifluoroacetic acid/1-[2-(4-aminopiperazin-1-yl)-2-oxoethyl]-1H-pyrrole-2,5-dione (1:1)

The title compound was prepared in 2 steps from tert-butyl piperazin-1-ylcarbamate and (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetic acid in analogy to intermediate L2.

HPLC (Method 11): R _t = 0.2 min;

LC-MS (Method 3): R _t = 0.25 min; MS (ESIpos): m/z = 239 (M+H) ⁺ .

Intermediate L15

Trifluoroacetic acid/N-(2-aminoethyl)-3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy]ethoxy}ethoxy)propionamide (1:1)

2.93 g (10.58 mmol) of tert-butyl 3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propanoate was dissolved in 100 ml of dioxane/water (1:1), and 3.28 g (21.15 mmol) of methyl 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate and saturated sodium bicarbonate solution were added until a pH of 6-7 was reached. The solution was stirred at room temperature for 30 minutes, after which the 1,4-dioxane was evaporated under vacuum. 200 ml of water was then added, and the mixture was extracted three times with 300 ml of ethyl acetate each time. The organic extracts were combined, dried over magnesium sulfate, and filtered. Concentration yielded tert-butyl 3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy]ethoxy}ethoxy)propanoate as a brown oil, which was subsequently dried under high vacuum.

HPLC (Method 11): R _t = 1.5 min;

LC-MS (Method 3): R _t = 0.88 min; MS (ESIpos): m/z = 375 (M+NH ₄ ) ⁺ .

This intermediate was converted to the title compound by standard methods (deprotection with TFA, coupling with tert-butyl (2-aminoethyl)carbamate and deprotection again with TFA).

HPLC (Method 11): R _t = 0.2 min;

LC-MS (Method 3): R _t = 0.25 min; MS (ESIpos): m/z = 344 (M+H) ⁺ .

Intermediate L16

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N ⁵ -carbamoyl-L-ornithine

535 mg (1.73 mmol) of commercially available 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione and 930 ml of N , N -diisopropylethylamine were added to a solution of 266 mg (1.33 mmol) of L-valyl-N ⁵ -carbamoyl-L-ornithine in 24 ml of DMF. The preparation was treated in an ultrasound bath for 24 hours and then concentrated to dryness under vacuum. The remaining residue was purified by preparative HPLC, and after concentration of the appropriate fractions and drying of the residue under high vacuum, 337 mg (50% of theory) of the title compound were obtained.

HPLC (Method 11): R _t = 0.4 min;

LC-MS (Method 3): R _t = 0.58 min; MS (ESIpos): m/z = 468 (M+H) ⁺ .

Intermediate L17

Trifluoroacetic acid/N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N ⁵ -carbamoyl-L-ornithine-L-lysine tert-butyl ester (1:1)

The title compound was prepared by initially coupling 172 mg (0.37 mmol) of intermediate L16 and 125 mg (0.37 mmol) of N6-(tert-butoxycarbonyl)-L-lysine tert-butyl ester hydrochloride (1:1) in the presence of EDC /HOBT and N,N-diisopropylethylamine, followed by deprotection of the amino group under mild conditions by stirring in 10% trifluoroacetic acid in DCM for 2 hours at room temperature. After lyophilization from acetonitrile/water, 194 mg (49% of theory) of the title compound was obtained over two steps.

HPLC (Method 11): R _t = 1.1 min;

LC-MS (Method 1): R _t = 0.58 min; MS (ESIpos): m/z = 652 (M+H) ⁺ .

Intermediate L18

Trifluoroacetic acid/β-alanyl-L-alanyl-N ⁵ -carbamoyl-N-[4-(2-methoxy-2-oxoethyl)phenyl]-L-ornithinamide (1:1)

The title compound was prepared from methyl (4-aminophenyl)acetate analogously to intermediate L7, successively according to the classical methods of peptide chemistry by coupling ^N2- (tert-butoxycarbonyl) ^-N5 -carbamoyl-L-ornithine in the presence of HATU, deprotection with TFA, coupling with N-(tert-butoxycarbonyl)-L-alanine 2,5-dioxopyrrolidin-1-yl ester, deprotection with TFA, coupling with N-(tert-butoxycarbonyl)-β-alanine 2,5-dioxopyrrolidin-1-yl ester and deprotection again with TFA, which gave 330 mg of the title compound.

HPLC (Method 11): R _t = 0.29 min;

LC-MS (Method 1): R _t = 0.41 min; MS (ESIpos): m/z = 465 (M+H) ⁺ .

Intermediate L19

Trifluoroacetic acid/L-alanyl-N ⁵ -carbamoyl-N-(4-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}phenyl)-L-ornithinamide (1:1)

The title compound was prepared from 1,4-phenylenediamine sequentially according to classical peptide chemistry methods. In the first step, 942 mg (8.72 mmol) of 1,4-phenylenediamine was monoacylated with 0.8 g (2.9 mmol) of N₂-(tert-butoxycarbonyl) ^-N₅ -carbamoyl-L-ornithine in the presence of HATU and N , N -diisopropylethylamine. In the second step, the second aniline amino group was similarly acylated with ( ^2,5 -dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetic acid in the presence of HATU and N , N -diisopropylethylamine. Deprotection with TFA, coupling with N-(tert-butoxycarbonyl)-L-alanine 2,5-dioxopyrrolidin-1-yl ester, and further deprotection with TFA subsequently yielded the title compound in three further synthetic steps, yielding 148 mg via this route.

LC-MS (Method 1): R _t = 0.21 min; MS (ESIpos): m/z = 474 (M+H) ⁺ .

LC-MS (Method 4): R _t = 0.2 min; MS (ESIpos): m/z = 474 (M+H) ⁺ .

Intermediate L20

Trifluoroacetic acid/L-valyl-N ⁵ -carbamoyl-N-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl]-L-ornithinamide (1:1)

The title compound was prepared analogously to intermediate L8 from commercially available 1-(4-aminophenyl)-1H-pyrrole-2,5-dione by sequential coupling with ^N2- (tert-butoxycarbonyl) ^-N5 -carbamoyl-L-ornithine in the presence of HATU, deprotection with TFA, coupling with N-(tert-butoxycarbonyl)-L-valine 2,5-dioxopyrrolidin-1-yl ester and deprotection again with TFA, yielding 171 mg of the title compound.

HPLC (Method 11): R _t = 0.28 min;

LC-MS (Method 1): R _t = 0.39 min; MS (ESIpos): m/z = 445 (M+H) ⁺ .

Intermediate L21

L-Valyl-N ⁶ -(tert-butoxycarbonyl)-N-[4-(2-methoxy-2-oxoethyl)phenyl]-L-lysinamide

The title compound was prepared according to classical methods of peptide chemistry from 0.42 g (2.56 mmol) of commercially available methyl (4-aminophenyl)acetate by sequential coupling with ^N₆- (tert-butoxycarbonyl) ^-N₂ -[(9H-fluoren-9-ylmethoxy)carbonyl]-L-lysine in the presence of HATU and N , N -diisopropylethylamine, deprotection with piperidine, coupling with N-[(benzyloxy)carbonyl]-L-valine 2,5-dioxopyrrolidin-1-yl ester in the presence of N , N -diisopropylethylamine, and subsequent hydrogenolytic cleavage of the benzyloxycarbonyl protecting group over 10% palladium on activated carbon. This yielded 360 mg (32% of theory over 4 steps) of the title compound.

HPLC (Method 11): R _t = 1.5 min;

LC-MS (Method 1): R _t = 0.73 min; MS (ESIpos): m/z = 493 (M+H) ⁺ .

Intermediate L22

Trifluoroacetic acid/N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-valyl-N-{4-[(2S)-2-amino-3-methoxy-3-oxopropyl]phenyl}-N ⁵ -carbamoyl-L-ornithinamide (1:1)

The title compound was prepared from N-(tert-butoxycarbonyl)-4-nitro-L-phenylalanine according to classical methods of peptide chemistry. 2.5 g (8.06 mmol) of this starting material were first converted into the cesium salt in a first step and then into the methyl ester using iodomethane in DMF.

The nitro group was then converted to an amino group by hydrogenolysis over 10% palladium-activated carbon in methanol.

The amino group thus generated is subsequently acylated with ^N5 -carbamoyl- ^N2 -[(9H-fluoren-9-ylmethoxy)carbonyl]-L-ornithine in the presence of HATU and N , N -diisopropylethylamine in DMF.In the next step, the Fmoc group is cleaved with piperidine in DMF.

Then, it is coupled with N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-valine in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, hydrated 1 -hydroxy-1H-benzotriazole, and N , N -diisopropylethylamine in DMF, and finally the tert-butoxycarbonyl group is cleaved with trifluoroacetic acid.

HPLC (Method 11): R _t = 1.6 min;

LC-MS (Method 1): R _t = 0.77 min; MS (ESIpos): m/z = 673 (M+H) ⁺ .

Intermediate L23

Trifluoroacetic acid/N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]-β-alaninamide (1:1)

The title compound was prepared from commercially available trifluoroacetic acid/1-(2-aminoethyl)-1H-pyrrole-2,5-dione (1:1) by coupling with N-(tert-butoxycarbonyl)-β-alanine in the presence of EDCI/HOBT and N , N -diisopropylethylamine and subsequent deprotection with trifluoroacetic acid.

HPLC (Method 11): R _t = 0.19 min.

Intermediate L24

Trifluoroacetic acid/1-amino-N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]cyclopropanecarboxamide (1:1)

114 mg (0.67 mmol) of commercially available 1-[(tert-butoxycarbonyl)amino]cyclopropanecarboxylic acid was dissolved in 25 ml of DCM, 110 mg (0.623 mmol) of commercially available trifluoroacetic acid/1-(2-aminoethyl)-1H-pyrrole-2,5-dione (1:1) and 395 μl of N , N -diisopropylethylamine were added, and the mixture was cooled to -10°C. 217 mg (0.793 mmol) of 2-bromo-1-ethylpyridinium tetrafluoroborate was then added, and the mixture was stirred at room temperature for 2 hours. The mixture was then diluted with ethyl acetate and extracted sequentially with 10% citric acid, saturated sodium bicarbonate solution, and saturated sodium chloride solution, followed by drying over magnesium sulfate and concentration. Drying under high vacuum yielded 152 mg of the protected intermediate.

These were then taken up in 10 ml of DCM and deprotected with 1 ml of trifluoroacetic acid. Lyophilization from acetonitrile/water yielded 158 mg (71% of theory over 2 steps) of the title compound.

HPLC (Method 11): R _t = 0.19 min.

LC-MS (Method 3): R _t = 0.98 min; MS (ESIpos): m/z = 224 (M+H) ⁺ .

Intermediate L25

N-[31-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatriacontanoyl]-L-valyl-L-alanine

31.4 mg (0.17 mmol) of valyl-L-alanine was dissolved in 3.0 ml of DMF, and 115.0 mg (0.17 mmol) of 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-{27-[(2,5-dioxopyrrolidin-1-yl)oxy]-27-oxo-3,6,9,12,15,18,21,24-octaoxaheptacosan-1-yl}propanamide and 33.7 mg (0.0.33 mmol) of triethylamine were added. The mixture was stirred overnight at room temperature. The reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 250x30; 10µm, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 74.1 mg (58% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.61 min; MS (ESIpos): m/z = 763 [M+H] ⁺ .

Intermediate L26

L-Valyl-N ⁶ -(tert-Butoxycarbonyl)-L-lysine

600.0 mg (1.58 mmol) of N ² -[(benzyloxy)carbonyl]-N ⁶ -(tert-butoxycarbonyl)-L-lysine was suspended in 25.0 ml of water/ethanol/THF (1:1:0.5), palladium on carbon (10%) was added, and the mixture was hydrogenated with hydrogen at standard pressure at room temperature for 5 hours. The catalyst was filtered off, and the solvent was evaporated under vacuum. The resulting compound was used in the next step without further purification.

LC-MS (Method 1): R _t = 0.42 min; MS (ESIpos): m/z = 247 [M+H] ⁺ .

Dissolve 180 mg (0.73 mmol) of N ⁶ -(tert-butoxycarbonyl)-L-lysine in 5.0 ml of DMF and add 74.0 mg (0.73 mmol) of triethylamine. Then, add 254.6 mg (0.73 mmol) of N-[(benzyloxy)carbonyl]-L-valine 2,5-dioxopyrrolidin-1-yl ester and 74.0 mg (0.73 mmol) of triethylamine. The reaction mixture is stirred at room temperature for 3.5 hours. The reaction solution is directly purified by preparative RP-HPLC (column: Reprosil 250x30; 10µm, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). Evaporate the solvent under vacuum, and the residue is dried under high vacuum. This gave 294.1 mg (76% of theory) of N-[(benzyloxy)carbonyl]-L-valyl-N ⁶ -(tert-butoxycarbonyl)-L-lysine.

LC-MS (Method 1): R _t = 0.97 min; MS (ESIpos): m/z = 480 [M+H] ⁺ .

Initially, 272.2 mg (0.57 mmol) of N-[(benzyloxy)carbonyl]-L-valyl-N ⁶ -(tert-butoxycarbonyl)-L-lysine was charged into 20.0 ml of ethyl acetate/ethanol/THF (1:1:1), and 27.2 mg of palladium-activated carbon were added. Hydrogenation was carried out at room temperature under standard pressure with hydrogen for 5 hours. The residue was filtered off using Celite ^(R) , and the filter cake was post-washed with ethyl acetate/ethanol/THF (1:1:1). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. The title compound (182 mg, 72% of theory) was used in the next reaction step without further purification.

LC-MS (Method 1): R _t = 0.53 min; MS (ESIpos): m/z = 346 [M+H] ⁺ .

Intermediate L27

N-[31-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatriacontane-1-yl]-L-valyl-N ⁶ -(tert-butoxycarbonyl)-L-lysine

30 mg (0.07 mmol) of L-valyl-N6-(tert-butoxycarbonyl)-L-lysine (Intermediate L26) and 46.1 mg (0.07 mmol) of 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-{27-[(2,5-dioxopyrrolidin-1-yl)oxy]-27-oxo-3,6,9,12,15,18,21,24-octaoxaheptacosan-1-yl}propanamide were initially charged in 1.5 ml of DMF, and 6.8 mg (0.07 mmol) of 4-methylmorpholine were added. The reaction solution was stirred at room temperature overnight. The reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 250x30; 10µm, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum and the residue was dried under high vacuum. This gave 55.6 mg (90% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.77 min; MS (ESIpos): m/z = 920 [M+H] ⁺ .

Intermediate L28

tert-Butyl 3-formyl-4-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)pyrrolidine-1-carboxylate

461.7 mg (1.15 mmol) of 1-tert-butyl 3-ethyl-4-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)pyrrolidine-1,3-dicarboxylate (this compound was prepared according to the literature procedure of WO 2006/066896) was initially charged in 5.0 mL of pure dichloromethane and cooled to -78°C. Then, 326.2 mg (2.29 mmol) of diisobutylaluminum hydride solution (1 M in THF) was slowly added dropwise, and the mixture was stirred at -78°C for 2 hours (monitored by thin-layer chromatography (petroleum ether/ethyl acetate = 3:1)). 1.3 g (4.59 mmol) of potassium sodium tartrate dissolved in 60 mL of water was added dropwise, and the reaction mixture was allowed to warm to room temperature. Ethyl acetate was added to the reaction mixture, and the aqueous phase was extracted three times with ethyl acetate. The combined organic phases were washed once with saturated NaCl solution and dried over magnesium sulfate. The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 629.0 mg of the title compound as a crude product which was used immediately in the next reaction step without further purification.

Intermediate L29

tert-Butyl 3-formyl-4-[({[2-(trimethylsilyl)ethoxy]carbonyl}amino)methyl]pyrrolidine-1-carboxylate

mixture of diastereomers

Initially, 807.1 mg (2.34 mmol) of tert-butyl 3-({[tert-butyl(dimethyl)silyl]oxy}methyl)-4-(hydroxymethyl)pyrrolidine-1-carboxylate (prepared according to the literature procedure of WO 2006/100036) was charged in 8.0 ml of dichloromethane, and 236.4 mg (2.34 mmol) of triethylamine was added. At 0°C, 267.6 mg (2.34 mmol) of methanesulfonyl chloride was added dropwise, and the reaction mixture was stirred at room temperature overnight. An additional 133.8 mg (1.17 mmol) of methanesulfonyl chloride and 118.2 mg (1.17 mmol) of triethylamine were added. The reaction mixture was stirred at room temperature overnight. The mixture was diluted with dichloromethane, and the organic phase was washed once with saturated sodium bicarbonate solution, once with 5% potassium bisulfate solution, and once with saturated NaCl solution. After drying over magnesium sulfate, the solvent was evaporated under vacuum, and the residue was purified on a Biotage Isolera (silica gel, 50 g SNAP column, flow rate 66 ml/min, cyclohexane/ethyl acetate). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 402.0 mg (41% of theory) of tert-butyl 3-({[tert-butyl(dimethyl)silyl]oxy}methyl)-4-{[(methylsulfonyl)oxy]methyl}pyrrolidine-1-carboxylate.

LC-MS (method 1): R _t = 1.38 min; MS (ESIpos): m/z = 424 [M+H] ⁺ .

400.0 mg (0.94 mmol) of tert-butyl 3-({[tert-butyl(dimethyl)silyl]oxy}methyl)-4-{[(methylsulfonyl)oxy]methyl}pyrrolidine-1-carboxylate were initially charged in 5.0 ml of DMF, and 98.2 mg (1.51 mmol) of sodium azide were added. The reaction mixture was stirred at 40°C for 10 hours. A further 30.7 mg (0.47 mmol) of sodium azide was then added and stirred at 40°C for a further 10 hours. Ethyl acetate was added, and the organic phase was repeatedly washed with water. After drying the organic phase over magnesium sulfate, the solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 309.5 mg (89% of theory) of tert-butyl 3-(azidomethyl)-4-({[tert-butyl(dimethyl)silyl]oxy}methyl)pyrrolidine-1-carboxylate. This compound was used in the next step of the synthesis without further purification.

LC-MS (Method 1): R _t = 1.50 min; MS (ESIpos): m/z = 371 [M+H] ⁺ .

250 mg (0.68 mmol) of tert-butyl 3-(azidomethyl)-4-({[tert-butyl(dimethyl)silyl]oxy}methyl)pyrrolidine-1-carboxylate was dissolved in 10.0 ml of ethyl acetate/ethanol (1:1), and 25.0 mg of palladium-activated carbon (10%) was added. Hydrogenation was carried out at room temperature under standard pressure with hydrogen for 8 hours. The reaction was filtered through Celite ^(R) , and the filter cake was washed thoroughly with ethyl acetate. The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 226.2 mg (82% of theory) of tert-butyl 3-(aminomethyl)-4-({[tert-butyl(dimethyl)silyl]oxy}methyl)pyrrolidine-1-carboxylate. This compound was used in the next step of the synthesis without further purification.

LC-MS (Method 1): R _t = 0.89 min; MS (ESIpos): m/z = 345 [M+H] ⁺ .

715.0 mg (2.08 mmol) of tert-butyl 3-(aminomethyl)-4-({[tert-butyl(dimethyl)silyl]oxy}methyl)pyrrolidine-1-carboxylate was dissolved in 15.0 ml of THF, and 2.28 ml (2.28 mmol) of TBAF solution (1 M in THF) was added. The reaction mixture was stirred at room temperature overnight. The solvent was evaporated under vacuum, and the residue (1.54 g) was used in the next step of the synthesis without further purification.

LC-MS (Method 1): R _t = 0.41 min; MS (ESIpos): m/z = 231 [M+H] ⁺ .

Initially, 1.54 g (4.88 mmol) of tert-butyl 3-(aminomethyl)-4-(hydroxymethyl)pyrrolidine-1-carboxylate was charged to 1,4-dioxane. 541.8 mg (4.88 mmol) of anhydrous calcium chloride and 488.6 mg (4.88 mmol) of calcium carbonate were added and stirred vigorously. Then, 592.8 mg (5.86 mmol) of triethylamine and 1.52 g (5.86 mmol) of 1-({[2-(trimethylsilyl)ethoxy]carbonyl}oxy)pyrrolidine-2,5-dione were added, and the reaction mixture was stirred at room temperature overnight. 644.9 mg (10.7 mmol) of HOAc and ethyl acetate were added. The organic phase was washed twice with water and once with saturated NaCl solution. After drying over magnesium sulfate, the solvent was evaporated under vacuum, and the residue was purified on silica gel (eluent: dichloromethane/methanol = 100:1). The solvent was evaporated under vacuum and the residue was dried under high vacuum. This gave 346.9 mg (19% of theory) of the compound tert-butyl 3-(hydroxymethyl)-4-[({[2-(trimethylsilyl)ethoxy]carbonyl}amino)methyl]pyrrolidine-1-carboxylate.

LC-MS (method 1): R _t = 1.08 min; MS (ESIpos): m/z = 375 [M+H] ⁺ .

804.0 mg (2.15 mmol) of tert-butyl 3-(hydroxymethyl)-4-[({[2-(trimethylsilyl)ethoxy]carbonyl}amino)methyl]pyrrolidine-1-carboxylate were initially charged in 20.0 ml of chloroform and 20.0 ml of 0.05 N potassium carbonate/0.05 N sodium bicarbonate solution (1:1). 59.7 mg (0.22 mmol) of tetra-n-butylammonium chloride, 429.9 mg (3.22 mmol) of N-chlorosuccinimide, and 33.5 mg (0.22 mmol) of TEMPO were then added, and the reaction mixture was stirred vigorously at room temperature overnight. The organic phase was separated and the solvent was removed under vacuum. The residue was purified on silica gel (eluent: cyclohexane/ethyl acetate = 3:1). This gave 517.0 mg (46% of theory) of the title compound.

LC-MS (method 1): R _t = 1.13 min; MS (ESIpos): m/z = 373 [M+H] ⁺ .

Intermediate L30

tert-Butyl 3-({[tert-butyl(dimethyl)silyl]oxy}methyl)-4-formylpyrrolidine-1-carboxylate

mixture of stereoisomers

Initially, 250.0 mg (0.72 mmol) of tert-butyl 3-({[tert-butyl(dimethyl)silyl]oxy}methyl)-4-(hydroxymethyl)pyrrolidine-1-carboxylate (prepared according to the literature procedure of WO2006/100036) was charged in 12.5 ml of dichloromethane/DMSO (4:1), and 219.6 mg (2.17 mmol) of triethylamine was added. At 2°C, 345.5 mg (2.17 mmol) of sulfur trioxide-pyridine complex was added portionwise, and the mixture was stirred at 2°C for 3 hours. An additional 345.5 mg (2.17 mmol) of sulfur trioxide-pyridine complex was added portionwise, and the mixture was stirred at room temperature for 17 hours. The reaction mixture was partitioned between dichloromethane and water. The aqueous phase was extracted three times with dichloromethane, and the combined organic phases were washed once with water and dried over magnesium sulfate. The solvent was evaporated under vacuum, and the residue was dried under high vacuum. The residue was used in the next step of the synthesis without further purification (thin layer chromatography: petroleum ether/ethyl acetate 7:3).

Intermediate L31

Di-tert-butyl {[(tert-butoxycarbonyl)amino]methyl}malonate

57.2 g (488.27 mmol) of tert-butyl carbamate, 51.2 ml (683.57 mmol) of 37% aqueous formaldehyde, and 25.9 g (244.13 mmol) of sodium carbonate were added to 600 ml of water. The mixture was warmed until a solution formed, then stirred at room temperature for 16 hours. The resulting suspension was extracted with 500 ml of dichloromethane, and the organic phase was separated, washed with saturated sodium chloride solution, and dried over sodium sulfate. The mixture was concentrated on a rotary evaporator, and the residue was dried under high vacuum to produce a crystalline solid. The residue was placed in 1000 ml of pure THF, and a mixture of 322 ml (3.414 mol) of acetic anhydride and 138 ml (1.707 mol) of pyridine was added dropwise at room temperature. The reaction mixture was stirred at room temperature for 16 hours, then concentrated on a rotary evaporator in a room temperature water bath. The residue was taken up in diethyl ether and washed three times with saturated sodium bicarbonate solution and once with saturated sodium chloride solution. The organic phase was dried over sodium sulfate and concentrated on a rotary evaporator, and the residue was dried under high vacuum for 2 days. The residue was placed in 2000 ml of pure THF, and 456 ml (456.52 mmol) of a 1 M solution of potassium tert-butoxide in THF was added under ice cooling. The mixture was stirred at 0°C for 20 minutes, followed by the dropwise addition of 100.8 g (456.52 mmol) of di-tert-butyl malonate dissolved in 200 ml of pure THF. The mixture was stirred at room temperature for 48 hours, after which water was added. The reaction mixture was concentrated on a rotary evaporator and placed in 500 ml of ethyl acetate. The mixture was washed with 500 ml of water and 100 ml of saturated sodium chloride solution, and the organic phase was dried over sodium sulfate. The organic phase was concentrated on a rotary evaporator, and the residue was dried under high vacuum. The residue was purified by filtration through silica gel (eluent: cyclohexane/ethyl acetate, gradient = 30:1 → 5:1). This yielded 37.07 g (22% of theory) of the target compound.

LC-MS (Method 6): R _t = 2.87 min; MS (ESIpos): m/z = 346 [M+H] ⁺ .

Intermediate L32

tert-Butyl [3-hydroxy-2-(hydroxymethyl)propyl]carbamate

37.0 g (107.11 mmol) of di-tert-butyl (acetoxymethyl)malonate was dissolved in 1000 ml of pure THF, and 535.5 ml (1071.10 mmol) of a 2M solution of lithium borohydride in THF was added dropwise under ice cooling. 19.3 ml (1071.10 mmol) of water was added dropwise, and the mixture was stirred at room temperature for 4.5 hours. The reaction mixture was concentrated on a rotary evaporator and dried under high vacuum. The residue was taken up in 1500 ml of ethyl acetate, 100 ml of water was added, and the mixture was stirred under ice cooling (slight exotherm) for 30 minutes. The organic phase was separated, and the aqueous phase was extracted twice with 500 ml of ethyl acetate. The organic phase was concentrated on a rotary evaporator, and the residue was dried under high vacuum. This yielded 20.7 g (94% of theory) of the target compound.

LC-MS (Method 6): R _t = 1.49 min; MS (EIpos): m/z = 106 [MC ₅ H ₈ O ₂ ] ⁺ .

Intermediate L33

tert-Butyl [3-{[tert-butyl(dimethyl)silyl]oxy}-2-(hydroxymethyl)propyl]carbamate

Dissolve 20.00 g (97.44 mmol) of tert-butyl [3-hydroxy-2-(hydroxymethyl)propyl]carbamate in 1000 ml of pure dichloromethane, and add 6.63 g (97.44 mmol) of imidazole and 16.16 g (107.18 mmol) of tert-butyl(chloro)dimethylsilane at room temperature. Stir at room temperature for 16 hours, and wash the reaction mixture with semiconcentrated sodium chloride solution. The aqueous phase is extracted with ethyl acetate, and the combined organic phases are dried over sodium sulfate, concentrated on a rotary evaporator, and dried under high vacuum. This yields 28.50 g (92% of theory) of the target compound.

¹ H NMR (400 MHz, DMSO-d ₆ ): δ [ppm] = 0.02 (s, 6H), 0.86 (s, 9H), 1.37(s, 9H), 1.58-1.73 (m, 1H), 2.91 (q, 2H), 3.33-3.36 [m, (2H, obscured)],3.53-3.58 (m, 2H), 6.65-6.72 (m, 1H).

Intermediate L34

tert-Butyl (3-{[tert-butyl(dimethyl)silyl]oxy}-2-formylpropyl)carbamate

12.65 g (39.591 mmol) of tert-butyl [3-{[tert-butyl(dimethyl)silyl]oxy}-2-(hydroxymethyl)propyl]carbamate was dissolved in 200 ml of dichloromethane, and 19.31 g (45.53 mmol) of Dess-Martin periodinane dissolved in 150 ml of dichloromethane was added dropwise at room temperature. The mixture was stirred at room temperature for 2 hours, then 250 ml of semiconcentrated sodium bicarbonate solution and 250 ml of 10% sodium thiosulfate solution were added and stirred for 20 minutes. The organic phase was separated, and the aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with 300 ml of water, dried over sodium sulfate, concentrated on a rotary evaporator, and dried under high vacuum. This yielded 11.35 g (90% of theory) of the target compound.

¹ H NMR (400 MHz, DMSO-d ₆ ): δ [ppm] = 0.02 (s, 6H), 0.84(s, 9H), 1.36(s, 9H), 1.48-1.51 (m, 1H), 3.08-3.32 [m, (1H, obscured)], 3.50-3.58 (m, 2H), 3.81-3.91 (m, 1H), 6.71 (t, 1H), 9.60 (d, 1H).

Intermediate L35

tert-Butyl (3-oxopropyl)carbamate

The title compound was prepared according to methods known in the literature (eg Jean Bastide et al . J. Med. Chem. 2003, 46 (16), 3536-3545).

Intermediate L36

N-[(Benzyloxy)carbonyl]-L-valyl-N5-carbamoyl-L-ornithine

100 mg (0.57 mmol) of N ⁵ -carbamoyl-L-ornithine was placed in 4.0 ml of DMF and 0.08 ml (0.57 mmol) of triethylamine was added. Then, 199.0 mg (0.57 mmol) of 2,5-dioxopyrrolidin-1-yl N-[(benzyloxy)carbonyl]-L-valine and 0.08 ml (0.57 mmol) of triethylamine were added. The mixture was stirred at room temperature for 48 hours. The reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 250x30; 10µm, flow rate: 50 ml/min, MeCN/water + 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 75.7 mg (33% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.69 min; MS (ESIpos): m/z = 409 [M+H] ⁺ .

Intermediate L37

L-Valyl-N ⁵ -carbamoyl-L-ornithine

75.7 mg (0.19 mmol) of intermediate L36 was suspended in 25 ml of water/ethanol/THF, 7.5 mg of palladium-activated carbon (10%) was added, and the mixture was hydrogenated with hydrogen at standard pressure at room temperature for 4.5 hours. The catalyst was filtered off, the reaction mixture was freed of solvent under vacuum, and dried under high vacuum. The residue was used in the next step without further purification. This yielded 64.9 mg (93% of theory) of the title compound.

LC-MS (Method 6): R _t = 0.25 min; MS (ESIpos): m/z = 275 [M+H] ⁺ .

Intermediate L38

N-[31-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatriacontanoyl-1-yl]-L-valyl-N ⁵ -carbamoyl-L-ornithine

Initially, 38.3 mg (0.14 mmol) of intermediate L37 was charged in 3.0 mL of DMF, followed by the addition of 96.4 mg (0.14 mmol) of 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-{27-[(2,5-dioxopyrrolidin-1-yl)oxy]-27-oxo-3,6,9,12,15,18,21,24-octaoxaheptacosan-1-yl}propanamide and 39.0 μL (0.28 mmol) of triethylamine. The mixture was stirred overnight at room temperature. 16.0 μL (0.28 mmol) of HOAc was then added to the reaction mixture, which was then purified directly by preparative RP-HPLC (column: Reprosil 250x30; 10µm, flow rate: 50 mL/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 58.9 mg (45% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.61 min; MS (ESIpos): m/z = 849 [M+H] ⁺ .

Intermediate L39

2-(Trimethylsilyl)ethyl (2-sulfanylethyl)carbamate

300 mg (2.64 mmol) of 2-aminoethanethiol hydrochloride (1:1) were initially charged in 3.0 ml of dichloromethane, and 668.0 mg (6.60 mmol) of triethylamine and 719.1 mg (2.77 mmol) of 1-({[2-(trimethylsilyl)ethoxy]carbonyl}oxy)pyrrolidine-2,5-dione were added. Stirring was carried out at room temperature for 2 days (monitored by thin-layer chromatography: dichloromethane/methanol = 100:1.5). Ethyl acetate was added to the reaction mixture, and the mixture was washed three times with water. The organic phase was washed twice with saturated NaCl solution and dried over magnesium sulfate. The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This compound was used in the next step of the synthesis without further purification.

Intermediate L40

N-[31-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatriacontane-1-yl]-L-valyl-N ⁶ -(tert-butoxycarbonyl)-L-lysine

600 mg (1.58 mmol) of ^N₂ -[(benzyloxy)carbonyl] ^-N₆- (tert-butoxycarbonyl)-L-lysine was hydrogenated in 25.0 ml of water/ethanol/THF (1:1:0.5) using palladium on carbon (10%) with hydrogen at room temperature under standard pressure. This compound, ^N₆- (tert-butoxycarbonyl)-L-lysine, was used in the next step of the synthesis without further purification.

LC-MS (Method 1): R _t = 0.99 min; MS (ESIpos): m/z = 247 [M+H] ⁺ .

Dissolve 180.0 g (0.73 mmol) of N ⁶ -(tert-Butoxycarbonyl)-L-lysine in 5.0 ml of DMF and add 74.0 mg (0.73 mmol) of triethylamine. Then, add 254.6 mg (0.73 mmol) of N-[(benzyloxy)carbonyl]-L-valine 2,5-dioxopyrrolidin-1-yl ester and 74.0 mg (0.73 mmol) of triethylamine. The reaction mixture is stirred at room temperature for 3.5 hours. The reaction mixture is directly purified by preparative RP-HPLC (column: Reprosil 250x30; 10µm, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). Evaporate the solvent under vacuum, and the residue is dried under high vacuum. This gave 294.1 mg (76% of theory) of the compound N-[(benzyloxy)carbonyl]-L-valyl-N ⁶ -(tert-butoxycarbonyl)-L-lysine.

LC-MS (Method 1): R _t = 0.97 min; MS (ESIpos): m/z = 480 [M+H] ⁺ .

272.2 mg (0.57 mmol) of N-[(benzyloxy)carbonyl]-L-valyl-N ⁶ -(tert-butoxycarbonyl)-L-lysine was dissolved in 20 ml of ethyl acetate/ethanol/THF (1:1:1), 27.2 mg of palladium-activated carbon was added, and the mixture was hydrogenated with hydrogen at standard pressure and room temperature. The mixture was filtered through Celite ^(R) , and the filter cake was washed thoroughly with ethyl acetate/ethanol/THF (1:1:1). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 182.0 mg (72% of theory) of the compound L-valyl-N ⁶ -(tert-butoxycarbonyl)-L-lysine.

LC-MS (Method 1): R _t = 0.53 min; MS (ESIpos): m/z = 346 [M+H] ⁺ .

30.0 mg (0.07 mmol) of L-valyl-N6-(tert-butoxycarbonyl)-L-lysine and 46.1 mg (0.07 mmol) of 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-{27-[(2,5-dioxopyrrolidin-1-yl)oxy]-27-oxo-3,6,9,12,15,18,21,24-octaoxaheptacosan-1-yl}propanamide were dissolved in 1.5 ml of DMF, and 6.8 mg (0.07 mmol) of 4-methylmorpholine was added. The reaction mixture was stirred at room temperature overnight. The reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 250x30; 10µm, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 55.6 mg (90% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.77 min; MS (ESIpos): m/z = 920 [M+H] ⁺ .

Intermediate L41

N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecanoyl]-L-valyl-N ⁶ -(tert-butoxycarbonyl)-L-lysine

600 mg (1.58 mmol) of ^N₂ -[(benzyloxy)carbonyl] ^-N₆- (tert-butoxycarbonyl)-L-lysine was hydrogenated in 25.0 ml of water/ethanol/THF (1:1:0.5) using palladium on carbon (10%) with hydrogen at room temperature under standard pressure. This compound, ^N₆- (tert-butoxycarbonyl)-L-lysine, was used in the next step of the synthesis without further purification.

LC-MS (Method 1): R _t = 0.99 min; MS (ESIpos): m/z = 247 [M+H] ⁺ .

Dissolve 180.0 g (0.73 mmol) of N ⁶ -(tert-Butoxycarbonyl)-L-lysine in 5.0 ml of DMF and add 74.0 mg (0.73 mmol) of triethylamine. Then, add 254.6 mg (0.73 mmol) of N-[(benzyloxy)carbonyl]-L-valine 2,5-dioxopyrrolidin-1-yl ester and 74.0 mg (0.73 mmol) of triethylamine. The reaction mixture is stirred at room temperature for 3.5 hours. The reaction mixture is then purified directly by preparative RP-HPLC (column: Reprosil 250x30; 10µm, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent is then evaporated under vacuum, and the residue is dried under high vacuum. This gave 294.1 mg (76% of theory) of the compound N-[(benzyloxy)carbonyl]-L-valyl-N ⁶ -(tert-butoxycarbonyl)-L-lysine.

LC-MS (Method 1): R _t = 0.97 min; MS (ESIpos): m/z = 480 [M+H] ⁺ .

272.2 mg (0.57 mmol) of N-[(benzyloxy)carbonyl]-L-valyl-N ⁶ -(tert-butoxycarbonyl)-L-lysine was dissolved in 20.0 ml of ethyl acetate/ethanol/THF (1:1:1), 27.2 mg of palladium-activated carbon was added, and the mixture was hydrogenated with hydrogen at standard pressure and room temperature. The mixture was filtered through Celite ^(R) , and the filter cake was washed thoroughly with ethyl acetate/ethanol/THF (1:1:1). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 182.0 mg (72% of theory) of the compound L-valyl-N ⁶ -(tert-butoxycarbonyl)-L-lysine.

LC-MS (Method 1): R _t = 0.53 min; MS (ESIpos): m/z = 346 [M+H] ⁺ .

30.0 mg (0.07 mmol) of L-valyl-N6-(tert-butoxycarbonyl)-L-lysine and 34.3 mg (0.07 mmol) of 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-{15-[(2,5-dioxopyrrolidin-1-yl)oxy]-15-oxo-3,6,9,12-tetraoxapentadecan-1-yl}propanamide were dissolved in 1.5 ml of DMF, and 6.8 mg (0.07 mmol) of 4-methylmorpholine were added. The reaction mixture was stirred at room temperature overnight. The reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 250x30; 10µm, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 40.6 mg (82% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.73 min; MS (ESIpos): m/z = 744 [M+H] ⁺ .

Intermediate L42

N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecanoyl]-L-valyl-N ⁵ -carbamoyl-L-ornithine

50.0 mg (0.18 mmol) of L-valyl-N ⁵ -carbamoyl-L-ornithine (Intermediate L37) was initially charged in DMF, and 93.6 mg (0.18 mmol) of 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-{15-[(2,5-dioxopyrrolidin-1-yl)oxy]-15-oxo-3,6,9,12-tetraoxapentadecan-1-yl}propanamide and 36.9 mg (0.37 mmol) of triethylamine were added. The reaction mixture was stirred at room temperature overnight. 21.9 mg (0.37 mmol) of HOAc was added, and the reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 250x30; 10µm, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 20.6 mg (14% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.55 min; MS (ESIpos): m/z = 673 [M+H] ⁺ .

Intermediate L43

N-[67-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-65-oxo-4,7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61-icosaoxa-64-azahexadecanoyl]-L-valyl-N ⁵ -carbamoyl-L-ornithine

11.3 mg (0.04 mmol) of L-valyl-N ⁵ -carbamoyl-L-ornithine (Intermediate L37) was initially charged in DMF and 50.0 mg (0.04 mmol) of 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-{63-[(2,5-dioxopyrrolidin-1-yl)oxy]-63-oxo-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60-icosaoxistatriacont-1-yl}propanamide and 8.3 mg (0.08 mmol) of triethylamine were added. The reaction mixture was stirred at room temperature overnight. 4.9 mg (0.08 mmol) of HOAc was added, and the reaction mixture was purified directly by preparative RP-HPLC (column: Reprosil 250x30; 10µm, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 15.8 mg (20% of theory) of the title compound.

LC-MS (Method 4): R _t = 0.94 min; MS (ESIpos): m/z = 1377 [M+H] ⁺ .

Intermediate L44

N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecanoyl]-L-valyl-L-alanine

73.3 mg (0.39 mmol) of L-valyl-L-alanine was dissolved in 7.0 ml of DMF, and 200.0 mg (0.39 mmol) of 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-{15-[(2,5-dioxopyrrolidin-1-yl)oxy]-15-oxo-3,6,9,12-tetraoxapentadecan-1-yl}propanamide and 78.8 mg (0.78 mmol) of triethylamine were added. The reaction mixture was stirred at room temperature overnight. The reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 250x30; 10µm, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 103.3 mg (45% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.58 min; MS (ESIpos): m/z = 587 [M+H] ⁺ .

Intermediate L45

tert-Butyl (2S)-2-[(tert-Butoxycarbonyl)amino]-4-oxobutanoate

2.00 g (7.26 mmol) of tert-butyl N-(tert-butoxycarbonyl)-L-homoserine ester was dissolved in 90 ml of dichloromethane, followed by the addition of 1.76 ml of pyridine and 4.62 g (10.90 mmol) of 1,1,1-triacetoxy-1λ ⁵ ,2-benzidoyl-3(1H)-one (Dess-Martin periodinane). The mixture was stirred at room temperature for 2 hours, then diluted with 200 ml of dichloromethane and extracted twice with 10% sodium thiosulfate solution, followed by two extractions with 5% citric acid and two extractions with saturated sodium bicarbonate solution. The organic phase was separated, dried over sodium sulfate, and then dried under vacuum. 100 ml of diethyl ether and cyclohexane (v/v = 1:1) were added to the residue, resulting in the formation of a white precipitate. This was filtered off with suction. The filtrate was concentrated on a rotary evaporator and dried under high vacuum to give 1.74 g (88% of theory) of the target compound as a light yellow oil.

LC-MS (Method 1): R _t = 0.85 min; MS (ESIpos): m/z = 274 [M+H] ⁺ .

¹ H NMR (400 MHz, DMSO-d ₆ ): δ [ppm] = 1.38 (s, 18H), 2.64-2.81 (m, 2H), 4.31-4.36 (m, 1H), 7.23 (d, 1H), 9.59 (s, 1H).

Intermediate L46

Trifluoroacetic acid/N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]-L-glutamic acid tert-butyl ester (1:1)

The title compound was prepared by first coupling 200 mg (0.79 mmol) of trifluoroacetic acid/1-(2-aminoethyl)-1H-pyrrole-2,5-dione (1:1) with 263 mg (0.87 mmol) of (4S) -5 -tert-butoxy-4-[(tert-butoxycarbonyl)amino]-5-oxopentanoic acid/trifluoroacetic acid (1:1) in the presence of EDC/HOBT and N,N-diisopropylethylamine, followed by deprotection of the amino group under mild conditions by stirring in 10% trifluoroacetic acid in DCM at room temperature for 1 hour. After lyophilization from acetonitrile/water, 85 mg (20% of theory) of the title compound were obtained over two steps.

LC-MS (Method 1): R _t = 0.37 min; MS (ESIpos): m/z = 326 [M+H] ⁺ .

Intermediate L47

Trifluoroacetic acid/β-alanyl-L-alanyl-N ⁵ -carbamoyl-N-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl]-L-ornithinamide (1:1)

The title compound was prepared by coupling intermediate L8 with N-(tert-butoxycarbonyl)-β-alanine 2,5-dioxopyrrolidin-1-yl ester and subsequent deprotection with TFA.

LC-MS (Method 3): R _t = 1.36 min; MS (ESIpos): m/z = 488 (M+H) ⁺ .

Intermediate L48

Trifluoroacetic acid/(1R,2S)-2-amino-N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]cyclopentanecarboxamide (1:1)

The title compound was prepared analogously to intermediate L2 from commercially available (1R,2S)-2-[(tert-butoxycarbonyl)amino]cyclopentanecarboxylic acid.

LC-MS (Method 3): R _t = 1.22 min; MS (ESIpos): m/z = 252 (M+H) ⁺ .

Intermediate L49

Trifluoroacetic acid/N-(bromoacetyl)-L-valyl-L-alanyl-L-lysine tert-butyl ester (1:1)

The title compound was prepared by first coupling commercially available bromoacetic anhydride with the partially protected peptide L-valyl- L -alanyl- ^N6- (tert-butoxycarbonyl)-L-lysine tert-butyl ester, prepared according to classical methods of peptide chemistry, in dichloromethane in the presence of N,N-diisopropylethylamine. This was followed by deprotection at the amino group under mild conditions by stirring in 10% trifluoroacetic acid in DCM at room temperature to give the title compound in 49% yield over two steps.

LC-MS (method 1): R _t = 1.09 min; MS (ESIpos): m/z = 593 and 595 (M+H) ⁺ .

Intermediate L50

Trifluoroacetic acid/(1S,3R)-3-amino-N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]cyclopentanecarboxamide (1:1)

The title compound was prepared from commercially available (1S,3R)-3-[(tert-butoxycarbonyl)amino]cyclopentanecarboxylic acid and also commercially available trifluoroacetic acid/1-(2-aminoethyl)-1H-pyrrole-2,5-dione (1:1) by coupling with HATU in the presence of N , N -diisopropylethylamine and subsequent deprotection with TFA.

HPLC (Method 11): R _t = 0.2 min;

LC-MS (Method 3): R _t = 0.88 min; MS (ESIpos): m/z = 252 (M+H) ⁺ .

Intermediate L51

Trifluoroacetic acid/(1R,3R)-3-amino-N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]cyclopentanecarboxamide (1:1)

The title compound was prepared from commercially available (1R,3R)-3-[(tert-butoxycarbonyl)amino]cyclopentanecarboxylic acid and also commercially available trifluoroacetic acid/1-(2-aminoethyl)-1H-pyrrole-2,5-dione (1:1) by coupling with HATU in the presence of N , N -diisopropylethylamine and subsequent deprotection with TFA.

LC-MS (Method 3): R _t = 0.98 min; MS (ESIpos): m/z = 250 (MH) ^- .

Intermediate L52

Trifluoroacetic acid/N-(2-aminoethyl)-2-bromoacetamide (1:1)

420 mg (2.62 mmol) of tert-butyl (2-aminoethyl)carbamate was placed in 50 ml of dichloromethane, and 817 mg (3.15 mmol) of bromoacetic anhydride and 913 μl (5.24 mmol) of N , N -diisopropylethylamine were added. The mixture was stirred at room temperature for 1 hour and then dried under vacuum. The residue was purified by preparative HPLC.

This produced 577 mg of the protected intermediate, which was then placed in 50 ml of dichloromethane and 10 ml of trifluoroacetic acid were added. After stirring at room temperature for 1 hour, the preparation was concentrated under vacuum and the residue was lyophilized from acetonitrile/water. This gave 705 mg (65% of theory) of the title compound.

LC-MS (Method 3): R _t = 0.34 min; MS (ESIpos): m/z = 181 and 183 (M+H) ⁺ .

Intermediate L53

Trifluoroacetic acid/(1S,3S)-3-amino-N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]cyclopentanecarboxamide (1:1)

The title compound was prepared from commercially available (1S,3S)-3-[(tert-butoxycarbonyl)amino]cyclopentanecarboxylic acid and likewise commercially available trifluoroacetic acid/1-(2-aminoethyl)-1H-pyrrole-2,5-dione (1:1) by coupling with HATU in the presence of N , N -diisopropylethylamine and subsequent deprotection with TFA.

HPLC (Method 11): R _t = 0.19 min;

LC-MS (Method 3): R _t = 0.88 min; MS (ESIpos): m/z = 250 (MH) ^- .

Intermediate L54

Trifluoroacetic acid/(1R,3S)-3-amino-N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]cyclopentanecarboxamide (1:1)

The title compound was prepared from commercially available (1R,3S)-3-[(tert-butoxycarbonyl)amino]cyclopentanecarboxylic acid and also commercially available trifluoroacetic acid/1-(2-aminoethyl)-1H-pyrrole-2,5-dione (1:1) by coupling with HATU in the presence of N,N -diisopropylethylamine and subsequent deprotection with TFA.

LC-MS (Method 3): R _t = 0.89 min; MS (ESIpos): m/z = 252 (M+H) ⁺ .

Intermediate L55

Trifluoroacetic acid/N ⁶ -D-alanyl-N ² -{N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-hexanoyl]-L-valyl-L-alanyl}-L-lysine tert-butyl ester (1:1)

The title compound was prepared by first coupling intermediate L6 with N-(tert-butoxycarbonyl)-D-alanine in the presence of HATU, followed by deprotection at the amino group under mild conditions by stirring in 5% trifluoroacetic acid in DCM at room temperature for 90 minutes.

HPLC (Method 11): R _t = 1.35 min;

LC-MS (Method 1): R _t = 0.67 min; MS (ESIpos): m/z = 637 (M+H) ⁺ .

Intermediate L56

Trifluoroacetic acid/N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-L-alanyl-N ⁶ -{[(1R,3S)-3-aminocyclopentyl]carbonyl}-L-lysine tert-butyl ester (1:1)

The title compound was prepared by first coupling intermediate L6 with (1R,3S)-3-[(tert-butoxycarbonyl)amino]cyclopentanecarboxylic acid in the presence of HATU, followed by deprotection at the amino group under mild conditions by stirring in 25% trifluoroacetic acid in DCM at room temperature for 15 minutes.

HPLC (Method 11): R _t = 1.4 min;

LC-MS (Method 1): R _t = 0.7 min; MS (ESIpos): m/z = 677 (M+H) ⁺ .

Intermediate L57

Methyl (2S)-4-oxo-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoate

500.0 mg (2.72 mmol) of L-aspartic acid methyl ester hydrochloride and 706.3 mg (2.72 mmol) of 2-(trimethylsilyl)ethyl 2,5-dioxopyrrolidine-1-carboxylate were initially charged in 5.0 ml of 1,4-dioxane, and 826.8 mg (8.17 mmol) of triethylamine were added. The reaction mixture was stirred at room temperature overnight. The reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 250x40; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was then evaporated under vacuum, and the residue was dried under high vacuum. This yielded 583.9 mg (74% of theory) of the compound (3S)-4-methoxy-4-oxo-3-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoic acid.

LC-MS (Method 1): R _t = 0.89 min; MS (ESIneg): m/z = 290 (MH) ^- .

592.9 mg of (3S)-4-methoxy-4-oxo-3-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butyric acid were initially charged in 10.0 ml of 1,2-dimethoxyethane, cooled to -15°C, and 205.8 mg (2.04 mmol) of 4-methylmorpholine and 277.9 mg (2.04 mmol) of isobutyl chloroformate were added. After 15 minutes, the precipitate was filtered off with suction and washed twice with 10.0 ml of 1,2-dimethoxyethane each time. The filtrate was cooled to -10°C, and 115.5 mg (3.05 mmol) of sodium borohydride dissolved in 10 ml of water was added with vigorous stirring. The phases were separated, and the organic phase was washed once with saturated sodium bicarbonate solution and once with saturated NaCl solution. The organic phase was dried over magnesium sulfate, the solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 515.9 mg (91% of theory) of the compound N-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-homoserine methyl ester.

LC-MS (Method 1): R _t = 0.87 min; MS (ESIpos): m/z = 278 (M+H) ⁺ .

Initially, 554.9 mg (2.00 mmol) of N-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-homoserine methyl ester was charged in 30.0 ml of dichloromethane, and 1.27 g (3.0 mmol) of Dess-Martin periodinane and 474.7 mg (6.00 mmol) of pyridine were added. Stirring was continued overnight at room temperature. After 4 hours, the preparation was diluted with dichloromethane, and the organic phase was washed three times each with ₁₀ % _{Na₂S₂O₃} solution, 10% _citric acid solution, and saturated sodium bicarbonate solution. The organic phase was dried over magnesium sulfate, and the solvent was evaporated under vacuum. This yielded 565.7 mg (97% of theory) of the title compound.

¹ H NMR (400 MHz, DMSO-d ₆ ): δ [ppm] = 0.03 (s, 9H), 0.91 (m, 2H), 2.70-2.79 (m, 1H), 2.88 (dd, 1H), 3.63 (s, 3H), 4.04 (m, 2H), 4.55 (m, 1H), 7.54(d, 1H), 9.60 (t, 1H).

Intermediate L58

2-(Trimethylsilyl)ethyl (3-oxopropyl)carbamate

434.4 mg (5.78 mmol) of 3-amino-1-propanol and 1.50 g (5.78 mmol) of 2-(trimethylsilyl)ethyl 2,5-dioxopyrrolidine-1-carboxylate were dissolved in 10.0 ml of dichloromethane, 585.3 mg (5.78 mmol) of triethylamine were added, and the mixture was stirred at room temperature overnight. The reaction mixture was diluted with dichloromethane, and the organic phase was washed with water and saturated sodium bicarbonate solution, then dried over magnesium sulfate. The solvent was evaporated under vacuum. The residue, 2-(trimethylsilyl)ethyl (3-hydroxypropyl)carbamate (996.4 mg, 79% of theory), was dried under high vacuum and used in the next step of the synthesis without further purification.

807.0 mg (3.68 mmol) of 2-(trimethylsilyl)ethyl (3-hydroxypropyl)carbamate was initially charged to 15.0 ml of chloroform and 15.0 ml of a 0.05 N potassium carbonate/0.05 N sodium bicarbonate solution (1:1). Then, 102.2 mg (0.37 mmol) of tetra-n-butylammonium chloride, 736.9 mg (5.52 mmol) of N-chlorosuccinimide, and 57.5 mg (0.37 mmol) of TEMPO were added, and the reaction mixture was stirred vigorously at room temperature overnight. The reaction mixture was diluted with dichloromethane, and the organic phase was washed with water and saturated NaCl solution. The organic phase was dried over magnesium sulfate, and the solvent was evaporated under vacuum. The residue was dried under high vacuum and used in the next step of the synthesis without further purification (890.3 mg).

Intermediate L59

Trifluoroacetic acid/1-{2-[2-(2-aminoethoxy)ethoxy]ethyl}-1H-pyrrole-2,5-dione (1:1)

300.0 mg (0.91 mmol) of tert-butyl (2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy]ethoxy}ethyl)carbamate were initially charged in dichloromethane, 4.2 g (36.54 mmol) of TFA were added, and the mixture was stirred at room temperature for 1 hour (DC monitoring: dichloromethane/methanol 10:1). The volatile components were evaporated under vacuum, and the residue was co-distilled four times with dichloromethane. The residue was dried under high vacuum and used in the next step of the synthesis without further purification.

LC-MS (Method 1): R _t = 0.19 min; MS (ESIpos): m/z = 229 (M+H) ⁺ .

Intermediate L60

6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl chloride

Dissolve 200.0 mg (0.95 mmol) of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid in 4.0 ml of dichloromethane and add 338.0 mg (2.84 mmol) of thionyl chloride. The reaction mixture is stirred at room temperature for 3 hours, followed by the addition of 1 drop of DMF. Stir for an additional hour. Evaporate the solvent under vacuum and co-distill three times with dichloromethane. The crude product is used in the next step of the synthesis without further purification.

Intermediate L61

Trifluoroacetic acid/N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-L-alanyl-L-lysine 2-(trimethylsilyl)ethyl ester (1:1)

First, the tripeptide derivative L-valyl-L-alanyl- ^N6- (tert-butoxycarbonyl)-L-lysine 2-(trimethylsilyl)ethyl ester was prepared from ^N2 -[(benzyloxy)carbonyl]-N6-(tert-butoxycarbonyl)-L-lysine according to classic peptide chemistry methods (esterification with ^2- (trimethylsilyl)ethanol using EDCI/DMAP, hydrogenolysis, coupling with N-[(benzyloxy)carbonyl]-L-valyl-L-alanine in the presence of HATU, and further hydrogenolysis. The title compound was prepared by coupling this partially protected peptide derivative with commercially available 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid in the presence of HATU and N , N -diisopropylethylamine. Deprotection at the amino group was then performed under mild conditions by stirring in 5% trifluoroacetic acid in DCM for 2.5 hours at room temperature, retaining the ester protecting group. Workup and purification by preparative HPLC yielded 438 mg of the title compound.

HPLC (Method 11): R _t = 1.69 min;

LC-MS (Method 1): R _t = 0.78 min; MS (ESIpos): m/z = 610 (M+H) ⁺ .

Intermediate L62

Trifluoroacetic acid/N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N ⁵ -carbamoyl-L-ornithine-L-lysine 2-(trimethylsilyl)ethyl ester (1:1)

First, ^N₆- (tert-butoxycarbonyl)-L-lysine 2-(trimethylsilyl)ethyl ester was prepared from ^N₂ -[(benzyloxy)carbonyl] ^-N₆- (tert-butoxycarbonyl)-L-lysine according to classical methods of peptide chemistry. 148 mg (0.43 mmol) of this intermediate was then coupled with 200 mg (0.43 mmol) of intermediate L16 in the presence of 195 mg (0.51 mmol) of HATU and 149 μL of N , N -diisopropylethylamine. After concentration and purification of the residue by preparative HPLC, the protected intermediate was taken up in 20 mL of DCM and the tert-butoxycarbonyl protecting group was removed by adding 2 mL of trifluoroacetic acid and stirring at room temperature for 1 hour. Concentration and lyophilization of the residue from acetonitrile/water yielded 254 mg (63% of theory over two steps).

HPLC (Method 11): R _t = 1.51 min;

LC-MS (Method 1): R _t = 0.68 min; MS (ESIpos): m/z = 696 (M+H) ⁺ .

Intermediate L63

(4S)-4-{[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}-5-oxo-5-[2-(trimethylsilyl)ethoxy]pentanoic acid

First, the tripeptide derivative (4S)-4-{[(2S)-2-{[(2S)-2-amino-3-methylbutanoyl]amino}propanoyl]amino}-5-oxo-5-[2-(trimethylsilyl)ethoxy]pentanoic acid was prepared from (2S)-5-(benzyloxy)-2-[(tert-butoxycarbonyl)amino]-5-oxopentanoic acid according to the classical methods of peptide chemistry (esterification with 2-(trimethylsilyl)ethanol using EDCI/DMAP, cleavage of the Boc protecting group with trifluoroacetic acid, coupling with N-[(benzyloxy)carbonyl]-L-valyl-L-alanine in the presence of HATU, and hydrogenolysis over 10% palladium-activated carbon in methanol). The title compound was prepared by coupling this partially protected peptide derivative with commercially available 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione. Workup and purification by preparative HPLC yielded 601 mg of the title compound.

LC-MS (Method 1): R _t = 0.96 min; MS (ESIpos): m/z = 611 (M+H) ⁺ .

Intermediate L64

(4S)-4-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}-5-oxo-5-[2-(trimethylsilyl)ethoxy]pentanoic acid

The title compound was prepared from (2S)-5-(benzyloxy)-2-[(tert-butoxycarbonyl)amino]-5-oxopentanoic acid according to classical methods of peptide chemistry (esterification with 2-(trimethylsilyl)ethanol using EDCI/DMAP, cleavage of the Boc protecting group with trifluoroacetic acid, cleavage of the benzyl ester over 10% palladium-activated carbon hydrogenolysis in methanol , and coupling with 1-{2-[(2,5-dioxopyrrolidin-1-yl)oxy]-2-oxoethyl}-1H-pyrrole-2,5-dione in the presence of N,N-diisopropylethylamine).

LC-MS (Method 1): R _t = 0.84 min; MS (ESIpos): m/z = 385 (M+H) ⁺ .

Intermediate L65

Trifluoroacetic acid/2-(trimethylsilyl)ethyl 3-{[(benzyloxy)carbonyl]amino}-L-alaninate (1:1)

The title compound was prepared from 3-{[(benzyloxy)carbonyl]amino}-N-(tert-butoxycarbonyl)-L-alanine according to classical peptide chemistry methods (esterification with 2-(trimethylsilyl)ethanol using EDCI/DMAP and cleavage of the Boc protecting group with trifluoroacetic acid). This yielded 373 mg (79% of theory over 2 steps) of the title compound.

LC-MS (Method 1): R _t = 0.72 min; MS (ESIpos): m/z = 339 (M+H) ⁺ .

Intermediate L66

(8S)-8-(2-Hydroxyethyl)-2,2-dimethyl-6,11-dioxo-5-oxa-7,10-diaza-2-sila-tetradec-14-oic acid methyl ester

Initially, 1000 mg (2.84 mmol) of (3S)-3-{[(benzyloxy)carbonyl]amino}-4-[(tert-butoxycarbonyl)amino]butyric acid was charged in 10.0 ml of 1,2-dimethoxyethane, and 344.4 mg (3.4 mmol) of 4-methylmorpholine and 504 mg (3.69 mmol) of isobutyl chloroformate were added. After stirring at room temperature for 10 minutes, the mixture was cooled to 5°C, and 161 mg (4.26 mmol) of sodium borohydride dissolved in 3 ml of water was added portionwise with vigorous stirring. After 1 hour, the same amount of sodium borohydride was added, and the mixture was then slowly warmed to room temperature. 170 ml of water was added, and the reaction was then extracted four times with 200 ml of ethyl acetate each time. The phases were separated, and the organic phase was washed once with citric acid and then with saturated sodium bicarbonate solution. The organic phase was dried over magnesium sulfate, the solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 760 mg (78% of theory) of the compound benzyl tert-butyl [(2S)-4-hydroxybutane-1,2-diyl]biscarbamate.

LC-MS (Method 1): R _t = 0.84 min; MS (ESIpos): m/z = 339 (M+H) ⁺ .

760 mg (2.16 mmol) of this intermediate dissolved in 13 ml of hydrogen chloride/dioxane were stirred at room temperature for 20 minutes. The preparation was then concentrated to 5 ml and diethyl ether was added. The precipitate was filtered off and lyophilized from acetonitrile/water 1:1.

The resulting product was dissolved in 132 ml of DMF and 345.5 mg (2.35 mmol) of 4-methoxy-4-oxobutanoic acid, 970 mg (2.55 mmol) of HATU, and 1025 μl of N , N -diisopropylethylamine were added. The mixture was stirred at room temperature for 5 minutes. The solvent was removed under vacuum, and the remaining residue was purified by preparative HPLC. The appropriate fractions were combined and the acetonitrile was evaporated under vacuum. The remaining aqueous phase was extracted twice with ethyl acetate, and the organic phase was then concentrated and dried under high vacuum.

The intermediate thus obtained is taken up in methanol and hydrogenated at room temperature under standard pressure of hydrogen over 10% palladium-activated carbon for 1 hour. The catalyst is then filtered off and the solvent is removed in vacuo.

247 mg of this deprotected compound was placed in 20 mL of DMF, and 352 mg (1.36 mmol) of 1-({[2-(trimethylsilyl)ethoxy]carbonyl}oxy)pyrrolidine-2,5-dione and 592 μL of N , N -diisopropylethylamine were added. The reaction mixture was stirred at room temperature for 1 hour, then concentrated, and the residue was purified by preparative HPLC. The solvent was then evaporated under vacuum, and the residue was dried under high vacuum. This yielded 218 mg of the title compound over these five reaction steps in an overall yield of 21%.

LC-MS (Method 1): R _t = 0.74 min; MS (ESIpos): m/z = 363 (M+H) ⁺ .

Intermediate L67

Trifluoroacetic acid/β-alanine 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl ester (1:1)

The title compound was prepared from 50 mg (0.354 mmol) of commercially available 1-(2-hydroxyethyl)-1H-pyrrole-2,5-dione by coupling with 134 mg (0.71 mmol) of N-(tert-butoxycarbonyl)-β-alanine in 10 ml of dichloromethane in the presence of 1.5 equivalents of EDCI and 0.1 equivalents of 4- N , N -dimethylaminopyridine and subsequent deprotection with trifluoroacetic acid.

Yield: 56 mg (48% of theoretical value over 2 steps)

LC-MS (Method 3): R _t = 1.15 min; MS (ESIpos): m/z = 213 (M+H) ⁺ .

Intermediate L68

Trifluoroacetic acid/N-(2-aminoethyl)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propionamide (1:1)

The title compound was prepared analogously to intermediate L1 according to classical methods of peptide chemistry from commercially available (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propionic acid and tert-butyl (2-aminoethyl)carbamate.

LC-MS (Method 1): R _t = 0.17 min; MS (ESIpos): m/z = 212 (M+H) ⁺ .

Intermediate L69

Trifluoroacetic acid/L-valyl-N ⁵ -carbamoyl-L-ornithine 1-[(benzyloxy)carbonyl]piperidin-4-yl ester (1:1)

The title compound was prepared by classical methods of peptide chemistry from commercially available benzyl 4-hydroxypiperidine-1-carboxylate by esterification with ^N2- (tert-butoxycarbonyl) ^-N5 -carbamoyl-L-ornithine using EDCI/DMAP, subsequent cleavage of the Boc with TFA, followed by coupling with N-[(tert-butoxy)carbonyl]-L-valine in the presence of HATU and N , N -diisopropylethylamine and finally again cleavage of the Boc with TFA.

LC-MS (Method 1): R _t = 0.62 min; MS (ESIpos): m/z = 492 (M+H) ⁺ .

Intermediate L70

9H-Fluoren-9-ylmethyl (3-oxopropyl)carbamate

Initially, 1000.0 mg (3.36 mmol) of 9H-fluoren-9-ylmethyl (3-hydroxypropyl)carbamate was charged into 15.0 ml of chloroform and 15.0 ml of a 0.05 N potassium carbonate/0.05 N sodium bicarbonate solution (1:1). Then, 93.5 mg (0.34 mmol) of tetra-n-butylammonium chloride, 673.6 mg (5.04 mmol) of N-chlorosuccinimide, and 52.5 mg (0.34 mmol) of TEMPO were added, and the reaction mixture was stirred vigorously at room temperature overnight. The reaction mixture was diluted with dichloromethane, and the organic phase was washed with water and saturated NaCl solution. The organic phase was dried over magnesium sulfate, and the solvent was evaporated under vacuum. The residue was dried under high vacuum and purified on silica gel (eluent: cyclohexane/ethyl acetate 3:1-1:1). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 589.4 mg (58% of theory) of the title compound.

LC-MS (Method 6): R _t = 2.15 min; MS (ESIpos): m/z = 296 (MH) ⁺ .

Intermediate L71

tert-Butyl [4-(chlorocarbonyl)phenyl]carbamate

Initially, 100.0 mg (0.42 mmol) of 4-[(tert-butoxycarbonyl)amino]benzoic acid was charged into 2.0 ml of dichloromethane, and 64.2 mg (0.51 mmol) of oxalyl chloride was added. The reaction mixture was stirred at room temperature for 30 minutes (monitored by DC: dichloromethane/methanol). An additional 192.6 mg (1.53 mmol) of oxalyl chloride and 1 drop of DMF were then added and stirred at room temperature for 1 hour. The solvent was evaporated under vacuum, and the residue was repeatedly co-distilled with dichloromethane. This residue was used in the next step of the synthesis without further purification.

Intermediate L72

(9S)-9-(Hydroxymethyl)-2,2-dimethyl-6,11-dioxo-5-oxa-7,10-diaza-2-sila-tetradec-14-oic acid benzyl ester

The title compound was prepared from commercially available benzyl tert-butyl [(2S)-3-hydroxypropane-1,2-diyl]biscarbamate according to classical methods of peptide chemistry by hydrogenolytic cleavage of the Z protecting group, subsequent coupling with 4-(benzyloxy)-4-oxobutanoic acid in the presence of EDCI/HOBT, followed by cleavage of the Boc protecting group with TFA and finally reaction with 1-({[2-(trimethylsilyl)ethoxy]carbonyl}oxy)pyrrolidine-2,5-dione in the presence of triethylamine.

LC-MS (Method 1): R _t = 0.94 min; MS (ESIpos): m/z = 425 [M+H] ⁺ .

Intermediate L73

N-(2-Aminoethyl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide

395.5 mg (1.87 mmol) of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid, 1.21 g (9.36 mmol) of N , N -diisopropylethylamine, and 854.3 mg (2.25 mmol) of HATU were added to a solution of 300 mg (1.87 mmol) of tert-butyl (2-aminoethyl)carbamate in 20 ml of dimethylformamide. The reaction mixture was stirred at room temperature for 5 minutes. After concentrating the mixture, the residue was taken up in DCM and washed with water. The organic phase was washed with brine, dried over magnesium sulfate, filtered, and concentrated. This yielded 408 mg (33%, 53% purity) of the title compound, which was used without further purification.

LC-MS (Method 1): R _t = 0.75 min; MS (ESIpos): m/z = 354 (M+H) ⁺ .

1 ml of TFA was added to a solution of tert-butyl (2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}ethyl) carbamate (408 mg, 0.365 mmol) in 7 ml of dichloromethane. The reaction mixture was stirred at room temperature for 0.5 hours. The reaction mixture was concentrated under vacuum, and the residue was co-distilled twice with dichloromethane. The residue was used further without further purification. This yielded 384 mg (94%, 57% purity) of the title compound.

LC-MS (Method 1): R _t = 0.26 min; MS (ESIpos): m/z = 254 (M+H) ⁺ .

Intermediate F1

N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-N-{2-[(4S)-2,5-dioxo-1,3-oxazolidin-4-yl]ethyl}-2-hydroxyacetamide

Under argon, 77 mg (0.14 mmol) of (2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butyric acid hydrochloride (1:1) (Intermediate C4) was dissolved in THF. 2 mg of activated carbon and 27.5 mg (0.14 mmol) of triphosgene were then added to the mixture, followed by stirring at 50°C for 10 minutes. After two additional additions of 27.5 mg of triphosgene each and stirring at room temperature for 30 minutes, the reaction was complete.

The activated carbon was filtered off through a syringe filter and the solvent was removed under vacuum. Acetonitrile was added to the preparation and concentrated again under vacuum. This gave 72 mg (95%) of the title compound, which was used for ADC coupling without further purification.

HPLC (Method 11): R _t = 2.28 min;

LC-MS (Method 1): R _t = 1.2 min; MS (ESIpos): m/z = 541 (M+H) ⁺ .

Intermediate F2

Trifluoroacetic acid/(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]butanamide (1:1)

55 mg (0.089 mmol) of (2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-2-[(tert-butoxycarbonyl)amino]butanoic acid (Intermediate C5) was placed in 12 mL of DMF, and 68 mg (0.268 mmol) of commercially available trifluoroacetic acid/1-(2-aminoethyl)-1H-pyrrole-2,5-dione (1:1) was added, along with 34.3 mg (0.18 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 27.4 mg (0.18 mmol) of hydrated 1-hydroxy- 1H -benzotriazole, and 47 μL (0.27 mmol) of N , N -diisopropylethylamine. The mixture was stirred at room temperature overnight. The solvent was removed in vacuo, leaving a residue which was purified by preparative HPLC. Concentration of the appropriate fractions yielded 20 mg (30% of theory) of the title compound after lyophilization from 1,4-dioxane.

HPLC (Method 11): R _t = 2.48 min;

LC-MS (Method 1): R _t = 1.29 min; MS (ESIpos): m/z = 737 (M+H) ⁺ .

20 mg (0.027 mmol) of this intermediate were dissolved in 5 ml of dichloromethane, 1 ml of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was then concentrated under vacuum, and the remaining residue was lyophilized from acetonitrile/water 1:1. This gave 19 mg (95% of theory) of the title compound.

HPLC (Method 11): R _t = 2.0 min;

LC-MS (Method 1): R _t = 0.9 min; MS (ESIpos): m/z = 637 (M+H) ⁺ .

¹ H NMR (500 MHz, DMSO-d ₆ ): d = 8.28 (t, 1H), 7.9-8.1 (m, 3H), 7.7-7.8 (m, 2H), 7.2-7.4 (m, 6H) 7.0-7.1 (m, 3H), 5.7 (s, 1H), 5.0 and 5.3 (2d, 2H), 4.08 and 4.25 (2d, 2H), 3.3-3.65 (m, 5H), 3.1-3.25 (m, 2H), 0.75 and 1.45 (2m, 2H), 0.9 (s, 9H).

Intermediate F3

Trifluoroacetic acid/N-[(3S)-3-amino-4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazine}-4-oxobutyl]-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide (1:1)

13 mg (0.021 mmol) of (2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-2-[(tert-butoxycarbonyl)amino]butanoic acid (Intermediate C5) was placed in 5 mL of DMF, followed by the addition of 33 mg (86 μmol) of O- (7-azabenzotriazol-1-yl) -N , N , N ', N' -tetramethyluronium hexafluorophosphate, 15 μL of N , N -diisopropylethylamine, and 22 mg (64 μmol) of commercially available 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide. The reaction mixture was stirred at room temperature for 1 hour. It was then concentrated under high vacuum, and the resulting residue was purified by preparative HPLC. This gave 9.5 mg (53% of theory) of the protected intermediate in the form of a colorless foam.

HPLC (Method 11): R _t = 2.1 min;

LC-MS (Method 1): R _t = 1.33 min; MS (ESIpos): m/z = 822 (M+H) ⁺ .

9.5 mg (0.011 mmol) of this intermediate were dissolved in 3 ml of dichloromethane, 1 ml of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was then concentrated under vacuum, and the remaining residue was lyophilized from acetonitrile/water 1:1. This gave 7 mg (70% of theory) of the title compound.

HPLC (Method 11): R _t = 1.75 min;

LC-MS (Method 1): R _t = 0.91 min; MS (ESIpos): m/z = 722 (M+H) ⁺ .

Intermediate F4

Trifluoroacetic acid/(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-N-(6-{[(2R)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propionyl]amino}hexyl)butanamide (1:1)

First, 30 mg (0.049 mmol) of (2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-2-[(tert-butoxycarbonyl)amino]butanoic acid (Intermediate C5) was coupled with trifluoroacetic acid/9H-fluoren-9-ylmethyl (6-aminohexyl)carbamate (1:1) in the presence of HATU, similar to Intermediate F3. The Fmoc protecting group was then removed with piperidine according to standard methods. This amine component was then coupled with (2R)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl chloride, which had been prepared from the free acid using thionyl chloride, in the presence of N , N -diisopropylethylamine. In the final step, the Boc protecting group was removed with trifluoroacetic acid in DCM. This gave 1.1 mg (3% over 4 steps) of the title compound.

HPLC (Method 11): R _t = 1.83 min;

LC-MS (Method 1): R _t = 0.96 min; MS (ESIpos): m/z = 764 (M+H) ⁺ .

Intermediate F5

Trifluoroacetic acid/(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(propionyl)amino]-N-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy]ethyl}butanamide (1:1)

The title compound was prepared analogously to intermediate F2 from 16 mg (0.026 mmol) of intermediate C5 and 8.5 mg (0.03 mmol) of intermediate L12. This gave 3 mg (13% of theory over 2 steps) of the title compound.

HPLC (Method 11): R _t = 2.0 min;

LC-MS (Method 1): R _t = 0.96 min; MS (ESIpos): m/z = 681 (M+H) ⁺ .

Intermediate F6

Trifluoroacetic acid/N-[(16S)-16-amino-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-12,15-dioxo-3,6,9-trioxa-13,14-diazaoctadec-18-yl]-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide (1:1)

8 mg (12.7 μmol) of trifluoroacetic acid/tert-butyl {(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-1-hydrazino-1-oxobutan-2-yl}carbamate (1:1) (Intermediate C6) were placed in 8 ml of DMF and 6 mg (19 μmol) of commercially available 3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy]ethoxy}ethoxy)propanoic acid, 5.8 mg (15 μmol) O -(7-azabenzotriazol-1-yl)- N , N , N ', N '-tetramethyluronium hexafluorophosphate (HATU) and 7 μl (38 μmol) N , N -diisopropylethylamine. The mixture was stirred at room temperature for 15 minutes. The solvent was then removed under vacuum, and the residue was taken up in acetonitrile/water 1:1 and adjusted to pH 2 with trifluoroacetic acid. Purification was performed by preparative HPLC. Combining the appropriate fractions, concentration, and lyophilization from acetonitrile/water 1:1 yielded 5 mg (41% of theory) of the Boc-protected intermediate. Cleavage of the Boc group with trifluoroacetic acid provided 4 mg (32% of theory over 2 steps) of the title compound.

HPLC (Method 11): R _t = 1.89 min;

LC-MS (Method 1): R _t = 0.89 min; MS (ESIpos): m/z = 812 (M+H) ⁺ .

Intermediate F7

Trifluoroacetic acid/(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-N-(2-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}ethyl)butanamide (1:1)

The title compound was prepared analogously to intermediate F2 from 25 mg (0.037 mmol) of intermediate C5 and 35 mg (0.112 mmol) of intermediate L1. This gave 14.4 mg (29% of theory over 2 steps) of the title compound.

HPLC (Method 11): R _t = 2.0 min;

LC-MS (Method 1): R _t = 0.9 min; MS (ESIpos): m/z = 694 (M+H) ⁺ .

¹ H NMR (500 MHz, DMSO-d ₆ ): d = 8.2 (m, 1H), 7.9-8.1 (m, 3H), 7.7-7.8 (m, 2H), 7.2-7.4 (m, 6H), 7.0-7.12 (m, 3H), 5.7 (s, 1H), 4.95 and 5.3 (2d, 2H), 4.1 and 4.25 (2d, 2H), 4.0 (s, 2H), 3.3-3.65 (m, 5H), 3.0-3.15 (m, 2H), 0.7 and 1.45 (2m, 2H), 0.88 (s, 9H).

Intermediate F8

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-L-alanyl-N6-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-L-lysine/trifluoroacetic acid (1:1)

The title compound was prepared analogously to intermediate F2 from 10 mg (0.016 mmol) of intermediate C5 and 13 mg (0.018 mmol) of intermediate L6. This gave 10 mg (49% of theory over 2 steps) of the title compound.

HPLC (Method 11): R _t = 1.97 min;

LC-MS (Method 1): R _t = 0.93 min; MS (ESIpos): m/z = 1006 (M+H) ⁺ .

Intermediate F9

Trifluoroacetic acid/N-{(3S)-3-amino-4-[1-(2-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino}-2-oxoethyl)hydrazine]-4-oxobutyl}-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide (1:1)

The title compound was prepared analogously to Intermediate F2 from 1.5 mg (0.002 mmol) of Intermediate C7 and 0.95 mg (0.004 mmol) of commercially available trifluoroacetic acid/1-(2-aminoethyl)-1H-pyrrole-2,5-dione (1:1). This gave 1.1 mg (52% of theory over 2 steps) of the title compound.

HPLC (Method 11): R _t = 1.9 min;

LC-MS (Method 1): R _t = 0.89 min; MS (ESIpos): m/z = 709 (M+H) ⁺ .

Intermediate F10

Trifluoroacetic acid/(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-N-(3-{[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl]amino}-3-oxopropyl)butanamide (1:1)

The title compound was prepared analogously to intermediate F2 from 14 mg (0.022 mmol) of intermediate C5 and 10 mg (0.025 mmol) of intermediate L5. This gave 4.5 mg (22% of theory over 2 steps) of the title compound.

HPLC (Method 11): R _t = 2.0 min;

LC-MS (Method 1): R _t = 0.93 min; MS (ESIpos): m/z = 756 (M+H) ⁺ .

Intermediate F11

Trifluoroacetic acid/N-[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]-4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)cyclohexanecarboxamide (1:1)

The title compound was prepared analogously to intermediate F2 from 12 mg (0.019 mmol) of intermediate C5 and 10 mg (0.021 mmol) of intermediate L4. This gave 7 mg (38% of theory over 2 steps) of the title compound.

HPLC (Method 11): R _t = 2.04 min;

LC-MS (Method 1): R _t = 0.93 min; MS (ESIpos): m/z = 776 (M+H) ⁺ .

Intermediate F12

Trifluoroacetic acid/(1R,2S)-2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)-N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]cyclopentanecarboxamide (1:1)

The title compound was prepared analogously to Intermediate F2 from 43 mg (0.071 mmol) of Intermediate C5 and 30 mg (0.071 mmol) of Intermediate L2. The diastereomers formed at the Boc-protected intermediate stage were separated from one another by preparative HPLC (Chromatorex C18-10/125 x 30/12 ml/min). The stereochemistry of the separated diastereomers could be assigned by comparison with the individual diastereomers prepared in a similar manner from commercially available (1S, 2R)-2-[(tert-butoxycarbonyl)amino]cyclopentanecarboxylic acid:

Fraction 1: 1S2R diastereomer

Yield: 13 mg (22%)

HPLC (Method 11): R _t = 2.52 min;

LC-MS (Method 1): R _t = 1.31 min; MS (ESIpos): m/z = 848 (M+H) ⁺ .

Fraction 2: 1R2S diastereomer

Yield: 10 mg (17%)

HPLC (Method 11): R _t = 2.56 min;

LC-MS (Method 1): R _t = 1.33 min; MS (ESIpos): m/z = 848 (M+H) ⁺ .

Deprotection of 10 mg (0.011 mmol) of the 1R2S diastereomer with TFA yielded 8 mg (75% of theory) of the title compound.

HPLC (Method 11): R _t = 2.04 min;

LC-MS (Method 1): R _t = 0.92 min; MS (ESIpos): m/z = 748 (M+H) ⁺ .

Intermediate F13

Trifluoroacetic acid/(1S,2R)-2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)-N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]cyclopentanecarboxamide (1:1)

The synthesis was carried out analogously to intermediate F13 and the title compound was obtained by deprotection of the 1S2R diastereomer.

HPLC (Method 11): R _t = 2.1 min;

LC-MS (Method 1): R _t = 0.94 min; MS (ESIpos): m/z = 748 (M+H) ⁺ .

Intermediate F14

Trifluoroacetic acid/N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl-L-valyl-N ⁵ -carbamoyl-N-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl]-L-ornithinamide (1:1)

The title compound was prepared by coupling 20 mg (0.028 mmol) of intermediate C5 and 18 mg (0.028 mmol) of intermediate L7 in the presence of HATU and subsequent deblocking with TFA. This gave 15 mg (49% of theory over 2 steps) of the title compound.

HPLC (Method 11): R _t = 1.97 min;

LC-MS (Method 1): R _t = 0.91 min; MS (ESIpos): m/z = 1012 (M+H) ⁺ .

Intermediate F15

Trifluoroacetic acid/N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl-L-alanyl-N5-carbamoyl-N-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl]-L-ornithinamide (1:1)

This intermediate was prepared by coupling 15 mg (0.022 mmol) of intermediate C8 and 14 mg (0.026 mmol) of intermediate L8 in the presence of HATU and subsequent deblocking with TFA. This gave 7 mg (27% of theory over 2 steps) of the title compound.

HPLC (Method 11): R _t = 1.85 min;

LC-MS (Method 1): R _t = 0.87 min; MS (ESIpos): m/z = 984 (M+H) ⁺ .

Intermediate F16

Trifluoroacetic acid/N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl-L-valyl-N5-carbamoyl-N-[4-(2-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino}-2-oxoethyl)phenyl]-L-ornithinamide (1:1)

First, 20 mg (0.03 mmol) of intermediate C3 were coupled analogously to intermediate F3 with trifluoroacetic acid/β-alanyl-L-valyl-N5-carbamoyl-N-[4-(2-methoxy-2-oxoethyl)phenyl]-L-ornithinamide (1:1) (intermediate L9) in the presence of HATU (yield: 15 mg (44% of theory)).

26 mg (0.023 mmol) of this intermediate, N-{(2S)-4-[(acetoxyacetyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino]-2-[(tert-butoxycarbonyl)amino]butyryl}-β-alanyl-L-valyl-N5-carbamoyl-N-[4-(2-methoxy-2-oxoethyl)phenyl]-L-ornithinamide, were dissolved in 5 ml of methanol, 1 ml of 2M lithium hydroxide solution was added, and the preparation was stirred at room temperature for 90 minutes. The solvent was then removed under vacuum, the residue was taken up in acetonitrile/water, and the preparation was adjusted to pH 2 using TFA. The product was then concentrated, and the residue was purified by preparative HPLC to yield 20 mg (81%) of the carboxyl compound.

This intermediate was then placed in 5 ml of DMF and coupled with 6 mg (0.022 mmol) of commercially available trifluoroacetic acid/1-(2-aminoethyl)-1H- pyrrole -2,5-dione (1:1) in the presence of 8.4 mg (0.022 mmol) of HATU and 16 μl of N ,N-diisopropylethylamine. Purification by preparative HPLC yielded 17 mg (76% of theory) of the protected intermediate. This was taken up in 3 ml of DCM and 1 ml of TFA was added. After stirring at room temperature for 45 minutes, the mixture was concentrated and the residue was taken up with diethyl ether. After suction filtration and drying under high vacuum, the residue yielded 15 mg (81%) of the title compound.

HPLC (Method 11): R _t = 1.9 min;

LC-MS (Method 1): R _t = 0.9 min; MS (ESIpos): m/z = 1097 (M+H) ⁺ .

Intermediate F17

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-L-alanyl-N ⁶ -{[(1R,2S)-2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butanoyl}amino)cyclopentyl]carbonyl}-L-lysine/trifluoroacetic acid (1:1)

The title compound was prepared analogously to Intermediate F12 from 6 mg (0.01 mmol) of Intermediate C5 and 8 mg (0.01 mmol) of Intermediate L10. The diastereomers formed at the Boc-protected intermediate stage were separated from one another by preparative HPLC (Chromatorex C18-10/125x30/12 ml/min). The stereochemistry of the separated diastereomers could be assigned by comparison with the individual diastereomers prepared in a similar manner from commercially available (1S, 2R)-2-[(tert-butoxycarbonyl)amino]cyclopentanecarboxylic acid:

Fraction 1: 1S2R diastereomer

Yield: 1 mg

HPLC (Method 11): R _t = 2.73 min;

LC-MS (Method 1): R _t = 1.37 min; MS (ESIpos): m/z = 1274 (M+H) ⁺ .

Fraction 2: 1R2S diastereomer

Yield: 0.7 mg

HPLC (Method 11): R _t = 2.81 min;

LC-MS (Method 1): R _t = 1.41 min; MS (ESIpos): m/z = 1274 (M+H) ⁺ .

Complete deprotection of 0.7 mg (0.001 mmol) of the 1R2S diastereomer was achieved by dissolving in 1 ml of DCM, adding 1 ml of TFA, and stirring at room temperature for 1 hour. Concentration under vacuum and lyophilization of the residue from acetonitrile/water yielded 0.68 mg (94% of theory) of the title compound.

HPLC (Method 11): R _t = 2.1 min;

LC-MS (Method 1): R _t = 0.97 min; MS (ESIpos): m/z = 1117 (M+H) ⁺ .

Intermediate F18

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-L-alanyl-N ⁶ -{[(1S,2R)-2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butanoyl}amino)cyclopentyl]carbonyl}-L-lysine/trifluoroacetic acid (1:1)

The title compound was prepared analogously to intermediate F17 from 8.9 mg (0.014 mmol) of intermediate C5 and 13 mg (0.014 mmol) of intermediate L11.

HPLC (Method 11): R _t = 2.2 min;

LC-MS (Method 1): R _t = 1.01 min; MS (ESIpos): m/z = 1117 (M+H) ⁺ .

Intermediate F19

N ⁶ -{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-N ² -[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-lysine/trifluoroacetic acid (1:1)

The title compound was prepared analogously to intermediate F2 by coupling 25 mg (0.041 mmol) of intermediate C5 with 55 mg (0.122 mmol) of intermediate L13 and subsequent deprotection.

HPLC (Method 11): R _t = 1.84 min;

LC-MS (Method 1): R _t = 0.88 min; MS (ESIpos): m/z = 780 (M+H) ⁺ .

Intermediate F20

Trifluoroacetic acid/(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-N-{4-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]piperazin-1-yl}butanamide (1:1)

The title compound was prepared analogously to intermediate F2 by coupling 10 mg (0.015 mmol) of intermediate C5 with 55 mg (0.122 mmol) of intermediate L14 and subsequent deprotection.

HPLC (Method 11): R _t = 1.9 min;

LC-MS (Method 1): R _t = 0.87 min; MS (ESIpos): m/z = 735 (M+H) ⁺ .

Intermediate F21

Trifluoroacetic acid/(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-N-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-12-oxo-3,6,9-trioxa-13-azapentadecan-15-yl]butanamide (1:1)

The title compound was prepared analogously to intermediate F2 by coupling 10 mg (0.015 mmol) of intermediate C5 with 7 mg (0.015 mmol) of intermediate L15 and subsequent deprotection.

LC-MS (Method 1): R _t = 0.89 min; MS (ESIpos): m/z = 840 (M+H) ⁺ .

Intermediate F22

Trifluoroacetic acid/N-[(3S)-3-amino-4-(1-{2-[(2-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}ethyl)amino]-2-oxoethyl}hydrazine)-4-oxobutyl]-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide (1:1)

The title compound was prepared analogously to intermediate F9 by coupling 13.7 mg (0.017 mmol) of intermediate C7 with 5.9 mg (0.017 mmol) of intermediate L1 and subsequent deprotection.

HPLC (Method 11): R _t = 2.3 min;

LC-MS (Method 1): R _t = 1.2 min; MS (ESIpos): m/z = 866 (M+H) ⁺ .

Intermediate F23

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N ⁵ -carbamoyl-L-ornithine-N ⁶ -{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butyryl}-L-lysine/trifluoroacetic acid (1:1)

The title compound was prepared analogously to intermediate F2 by coupling 10 mg (0.016 mmol) of intermediate C5 with 16.8 mg (0.016 mmol) of intermediate L17 in the presence of EDC/HOBT and N , N -diisopropylethylamine and subsequent deprotection.

HPLC (Method 11): R _t = 1.9 min;

LC-MS (Method 1): R _t = 0.9 min; MS (ESIpos): m/z = 1092 (M+H) ⁺ .

Intermediate F24

Trifluoroacetic acid/N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl-L-alanyl-N ⁵ -carbamoyl-N-[4-(2-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino}-2-oxoethyl)phenyl]-L-ornithinamide (1:1)

The title compound was prepared analogously to intermediate F16:

First, 30 mg (0.046 mmol) of intermediate C3 were coupled with intermediate L18 in analogy to intermediate F3 in the presence of HATU (yield: 25 mg (47% of theory)).

27 mg (0.024 mmol) of this intermediate was dissolved in 5 ml of methanol, 1 ml of 2M lithium hydroxide solution was added, and the preparation was stirred at room temperature for 30 minutes to cleave both the methyl ester and the acetyl groups. The solvent was then removed under vacuum, the residue was taken up in acetonitrile/water, and the preparation was adjusted to pH 2 using TFA. The mixture was then concentrated, and after purification of the residue by preparative HPLC, 15 mg (58%) of the carboxyl compound was obtained.

This intermediate was then placed in 3 ml of DMF and coupled with 4.4 mg (0.017 mmol) of commercially available trifluoroacetic acid/1-(2-aminoethyl)-1H- pyrrole -2,5-dione (1:1) in the presence of 6.5 mg (0.017 mmol) of HATU and 12 μl of N ,N-diisopropylethylamine. Purification by preparative HPLC yielded 12 mg (72% of theory) of the protected intermediate. This was taken up in 2 ml of DCM and 1 ml of TFA was added. After stirring at room temperature for 30 minutes, the mixture was concentrated and lyophilized from acetonitrile/water (1:1). Drying the residue under high vacuum provided 11 mg (91%) of the title compound.

LC-MS (Method 1): R _t = 0.83 min; MS (ESIpos): m/z = 1069 (M+H) ⁺ .

Intermediate F25

Trifluoroacetic acid/N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl-L-alanyl-N ⁵ -carbamoyl-N-(4-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}phenyl)-L-ornithinamide (1:1)

This intermediate was prepared by coupling 9.6 mg (0.014 mmol) of intermediate C8 with 10.8 mg (0.015 mmol) of intermediate L19 in the presence of 6.4 mg (0.017 mmol) of HATU and 72 μl of N , N -diisopropylethylamine and subsequent deblocking with TFA. This gave 5 mg (31% of theory over 2 steps) of the title compound.

HPLC (Method 11): R _t = 1.8 min;

LC-MS (Method 1): R _t = 0.85 min; MS (ESIpos): m/z = 1041 (M+H) ⁺ .

Intermediate F26

Trifluoroacetic acid/N-{(15S,19R)-15-amino-19-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-18-glycoloyl-20,20-dimethyl-14-oxo-4,7,10-trioxa-13,18-diazaheneicosane-1-yl}-L-valyl-N ⁵ -carbamoyl-N-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl]-L-ornithinamide (1:1)

This intermediate was prepared by coupling 16.4 mg (0.02 mmol) of intermediate C9 with 11.2 mg (0.02 mmol) of intermediate L20 in the presence of 8 mg (0.04 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 6 mg (0.04 mmol) of hydrated 1-hydroxy- 1H -benzotriazole, and 11 μl (0.06 mmol) of N , N -diisopropylethylamine, and subsequent deblocking with TFA. This gave 10 mg (37% of theory over 2 steps) of the title compound.

HPLC (Method 11): R _t = 2.0 min;

LC-MS (Method 1): R _t = 0.91 min; MS (ESIpos): m/z = 1144 (M+H) ⁺ .

Intermediate F27

Trifluoroacetic acid/N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl-L-valyl-N-[4-(2-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino}-2-oxoethyl)phenyl]-L-lysinamide (2:1)

This intermediate is prepared in 4 steps:

In the first step, 20 mg (0.028 mmol) of intermediate C8 was coupled with 16.7 mg (0.031 mmol) of intermediate L21 in 5 mL of DMF in the presence of 11 mg (0.057 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 8.7 mg (0.057 mmol) of 1-hydroxy- 1H -benzotriazole hydrate, and 15 μl (0.085 mmol) of N , N -diisopropylethylamine. After stirring at room temperature for 4 days, the preparation was concentrated, and the product was purified by preparative HPLC. Yield: 18 mg (54.5% of theory).

18 mg (0.016 mmol) of this intermediate was dissolved in 4 ml of methanol, 194 μl of 2M lithium hydroxide solution was added, and the mixture was stirred at room temperature overnight. An additional 116 μl of lithium hydroxide solution was then added, and the mixture was stirred at room temperature for another 4 hours. The solvent was then removed under vacuum, the residue was taken up in water, and the pH of the mixture was adjusted to 5 with 5% citric acid. The mixture was extracted twice with dichloromethane, and the organic phase was dried over sodium sulfate. The organic phase was then filtered and concentrated, and the residue was dried under high vacuum. This yielded 10.5 mg (58%) of the carboxyl compound.

10.5 mg (0.009 mmol) of this intermediate was then placed in 4 ml of DMF and coupled with 3 mg (0.012 mmol) of commercially available trifluoroacetic acid/1-(2-aminoethyl)-1H-pyrrole-2,5-dione (1:1) in the presence of 3.5 mg (0.018 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 2.8 mg (0.018 mmol) of 1-hydroxy- 1H -benzotriazole hydrate, and 6 μl of N , N -diisopropylethylamine. After stirring overnight, the same amount of coupling reagent was added, and the mixture was stirred at room temperature for a further 3 days. The mixture was then concentrated, and the product was purified by preparative HPLC. Yield: 6 mg (52% of theory).

6 mg (0.005 mmol) of this intermediate were then deprotected in 3 ml of DCM with 1 ml of trifluoroacetic acid to yield 6 mg (83% of theory) of the title compound after lyophilization from acetonitrile/water.

HPLC (Method 11): R _t = 1.84 min;

LC-MS (Method 4): R _t = 0.93 min; MS (ESIpos): m/z = 1068 (M+H) ⁺ .

Intermediate F28

N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl-4-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N ⁵ -carbamoyl-L-ornithine}amino)-L-phenylalanine/trifluoroacetic acid (1:1)

This intermediate is prepared in 5 steps:

In the first step, 40 mg (0.058 mmol) of intermediate C8 was coupled with 46 mg (0.058 mmol) of intermediate L22 in the presence of 44.3 mg (0.117 mmol) of HATU and 30 μL of N , N -diisopropylethylamine. After stirring at room temperature for 1 hour, the preparation was concentrated, and the product was purified by preparative HPLC. Yield: 53 mg (62.5% of theory).

In the next step, the Fmoc group was cleaved with 0.6 ml of piperidine in 3 ml of DMF. After stirring at room temperature for 1 hour, the preparation was concentrated and the product was purified by preparative HPLC. Yield: 42 mg (82% of theory).

To cleave the methyl ester, 42 mg (0.033 mmol) of this intermediate were dissolved in 2 ml of THF and 1 ml of water, 330 μl of 2M lithium hydroxide solution were added and the preparation was stirred at room temperature for 1 hour.

The preparation was then neutralized with TFA and concentrated, and the residue was purified by preparative HPLC. Drying under high vacuum gave 32 mg (78%) of the carboxylic compound.

32 mg (0.026 mmol) of this intermediate was then coupled with 14.6 mg (0.047 mmol) of commercially available 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl } -1H-pyrrole-2,5-dione in 2.3 ml of DMF in the presence of 18 μl of N,N-diisopropylethylamine. After 4 hours of sonication, the preparation was concentrated, and the product was purified by preparative HPLC. Yield: 20.4 mg (60% of theory).

In the final step, 20.4 mg (0.016 mmol) of this intermediate were deprotected with trifluoroacetic acid in DCM to yield 20 mg (85% of theory) of the title compound after lyophilization from acetonitrile/water.

HPLC (Method 11): R _t = 1.9 min;

LC-MS (Method 5): R _t = 2.84 min; MS (ESIpos): m/z = 1197 (M+H) ⁺ .

Intermediate F29

N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl-4-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-L-alanyl}amino)-L-phenylalanine/trifluoroacetic acid (1:1)

The title compound was prepared analogously to intermediate F28.

HPLC (Method 11): R _t = 2.0 min;

LC-MS (Method 1): R _t = 0.92 min; MS (ESIpos): m/z = 1111 (M+H) ⁺ .

Intermediate F30

Trifluoroacetic acid/(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-N-{2-[(3-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino}-3-oxopropyl)sulfinyl]ethyl}butanamide (1:1)

This intermediate is prepared in 4 steps:

In the first step, 37.5 mg (0.055 mmol) of intermediate C3 was coupled with 15 mg (0.066 mmol) of commercially available methyl 3-[(2-aminoethyl)sulfanyl]propanoate hydrochloride (1:1) in DMF in the presence of 25 mg (0.066 mmol) of HATU and 29 μl of N , N -diisopropylethylamine. After stirring at room temperature for 15 minutes, the coupling reagent was added. The preparation was stirred at room temperature for a further 15 minutes, then concentrated, and the product was purified by preparative HPLC. Yield: 21 mg (48% of theory).

LC-MS (Method 1): R _t = 1.41 min; MS (ESIpos): m/z = 802 (M+H) ⁺ .

To cleave the methyl ester, 21 mg (0.026 mmol) of this intermediate was dissolved in 5 mL of methanol, 655 μL of 2M lithium hydroxide solution was added, and the mixture was stirred at room temperature overnight. During this time, partial oxidation of the sulfur occurred. The mixture was concentrated, and the residue was taken up in water and adjusted to pH 3 with acetic acid. It was extracted twice with 50 mL of ethyl acetate, and the organic phase was dried over magnesium sulfate, filtered, and concentrated. The residue was dried under high vacuum, and the resulting mixture was used without further purification in the next step by coupling with 8.4 mg (0.033 mmol) of commercially available trifluoroacetic acid/1-(2-aminoethyl)-1H-pyrrole-2,5-dione (1:1) in the presence of 11.6 mg (0.031 mmol) of HATU and 22 μL of N , N -diisopropylethylamine. The mixture was stirred at room temperature for 5 minutes and then concentrated. The residue was taken up in ethyl acetate, and the solution was extracted with 5% citric acid and then water. The organic phase was then dried over magnesium sulfate, filtered, and concentrated. The residue was dried under high vacuum and the resulting mixture was used in the next step without further purification.

Then 22 milligrams of this crude material are dissolved among 2 milliliters of DCM and use 0.5 milliliter of trifluoroacetic acid deprotection.After at room temperature stirring 10 minutes, concentrate this preparation, residue is purified by preparative HPLC.After the drying under high vacuum, produce 2.1 milligrams of title compounds.

HPLC (Method 11): R _t = 1.8 min;

LC-MS (Method 1): R _t = 0.83 min; MS (ESIpos): m/z = 784 (M+H) ⁺ .

Intermediate F31

Trifluoroacetic acid/N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-(3-{[2-{(1R)-1-[(3-aminopropyl)(ethanolyl)amino]-2,2-dimethylpropyl}-4-(2,5-difluorophenyl)-1H-imidazol-1-yl]methyl}phenyl)-L-alaninamide (1:1)

This intermediate was synthesized in 6 steps from intermediate C10 using classical methods of peptide chemistry.

In the first step, 42 mg (0.066 mmol) of intermediate C10 was coupled with 20.7 mg (0.066 mmol) of N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-alanine in 5 mL of DMF in the presence of 100 mg (0.266 mmol) of HATU and 46 μL of N , N -diisopropylethylamine. The preparation was stirred at room temperature overnight, and the product was purified by preparative HPLC. This yielded 16 mg (27% of theory) of the N-acylated compound and 9 mg (12% of theory) of the N-,O-bisacylated compound.

The N-acylated compound was deprotected with piperidine in DMF. The diacylated compound was treated with piperidine and an aqueous solution of methylamine in ethanol. In both cases, tert-butyl (3-{[(1R)-1-{1-[3-(L-alanylamino)benzyl]-4-(2,5-difluorophenyl)-1H-imidazol-2-yl}-2,2-dimethylpropyl](ethanolyl)amino}propyl)carbamate was formed and 13 mg were isolated after purification by preparative HPLC with a purity of 95%.

LC-MS (Method 1): R _t = 0.95 min; MS (ESIpos): m/z = 657 (M+H) ⁺ .

13 mg (0.019 mmol) of this intermediate was coupled with 9.1 mg (0.021 mmol) of N-[(9H-fluoren-9-ylmethoxy)carbonyl] -L - valine 2,5-dioxopyrrolidin-1-yl ester in 2 mL of DMF in the presence of 7 μL of N,N-diisopropylethylamine. After stirring at room temperature for 20 hours, the product was concentrated and the residue purified by preparative HPLC. Lyophilization from 1,4-dioxane/water yielded 10 mg (54% of theory).

The Fmoc protecting group was subsequently cleaved with piperidine in DMF to yield 9 mg (quantitative) of the partially deprotected intermediate.

9 mg (0.01 mmol) of this intermediate was then coupled with 3.2 mg (0.01 mmol) of commercially available 1-{6-[(2,5-dioxopyrrolidin-1 - yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione in 2 mL of DMF in the presence of 5 μL of N,N-diisopropylethylamine. After stirring overnight at room temperature, the preparation was concentrated, and the product was purified by preparative HPLC. Lyophilization from acetonitrile/water and a few drops of 1,4-dioxane afforded 3 mg (32% of theory), which was deprotected in a final step using 0.5 mL of trifluoroacetic acid in 2 mL of DCM. Lyophilization from acetonitrile/water yielded 3.8 mg (quantitative) of the title compound.

HPLC (Method 11): R _t = 1.9 min;

LC-MS (Method 1): R _t = 0.89 min; MS (ESIpos): m/z = 849 (M+H) ⁺ .

Intermediate F32

Trifluoroacetic acid/(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-N-(3-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino}-3-oxopropyl)butanamide (1:1)

This intermediate was prepared by coupling 15 mg (0.041 mmol) of intermediate C5 with 16.8 mg (0.027 mmol) of intermediate L23 in the presence of 10.5 mg (0.055 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 8.4 mg (0.055 mmol) of hydrated 1-hydroxy- 1H -benzotriazole and 14 μl (0.08 mmol) of N , N -diisopropylethylamine, and subsequent deblocking with TFA. This gave 3.4 mg (15% of theory over 2 steps) of the title compound.

HPLC (Method 11): R _t = 1.9 min;

LC-MS (Method 1): R _t = 0.85 min; MS (ESIpos): m/z = 708 (M+H) ⁺ .

Intermediate F33

Trifluoroacetic acid/(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-N-{2-[(bromoacetyl)amino]ethyl}butanamide (1:1)

The synthesis of this intermediate began in the first step by coupling 50 mg (0.075 mmol) of intermediate C3 with 26.2 mg (0.082 mmol) of 9H-fluoren-9-ylmethyl (2-aminoethyl)carbamate hydrochloride (1:1) in the presence of 28.7 mg (0.15 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 22.9 mg (0.15 mmol) of 1-hydroxy- 1H -benzotriazole hydrate, and 39 μl of N , N -diisopropylethylamine. After stirring at room temperature for 18 hours, the mixture was concentrated and the residue was purified by preparative HPLC. This yielded 45 mg (65% of theory) of this intermediate.

LC-MS (Method 1): R _t = 1.51 min; MS (ESIpos): m/z = 921 (M+H) ⁺ .

45 mg (0.049 mmol) of this intermediate was dissolved in 10 ml of ethanol, and 176 μl of a 40% aqueous methylamine solution was added. The mixture was stirred at 50°C, and the same amount of methylamine solution was added after 6 and 9 hours. After stirring at 50°C for a further 14 hours, an additional 700 μl of methylamine solution was added, and after stirring for a further 20 hours, the mixture was concentrated. The residue was taken up in DCM and washed with water. The organic phase was concentrated, and the residue was purified by preparative HPLC. Concentration of the appropriate fractions and drying of the residue under high vacuum yielded 32 mg (99% of theory) of tert-butyl {(2S)-1-[(2-aminoethyl)amino]-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-1-oxobutan-2-yl}carbamate.

LC-MS (Method 1): R _t = 0.95 min; MS (ESIpos): m/z = 657 (M+H) ⁺ .

8.3 mg (0.013 mmol) of this intermediate was dissolved in 4 ml of dichloromethane, and 3.3 mg (0.013 mmol) of bromoacetic anhydride and 2 μl of N , N -diisopropylethylamine were added. After stirring at room temperature for 1 hour, the mixture was concentrated, and the residue was purified by preparative HPLC. Appropriate fractions were concentrated, and the residue was lyophilized from acetonitrile/water. The residue was then taken up in 1 ml of dichloromethane and deprotected with 0.5 ml of trifluoroacetic acid. Concentration and lyophilization from acetonitrile/water yielded 1.1 mg (9% of theory over 2 steps) of the title compound.

HPLC (Method 11): R _t = 1.9 min;

LC-MS (Method 1): R _t = 0.88 min; MS (ESIpos): m/z = 677/679 (M+H) ⁺ .

Intermediate F34

Trifluoroacetic acid/N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-L-alanyl-N ⁵ -carbamoyl-N-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl]-L-ornithinamide (1:1)

This intermediate was prepared by coupling 14 mg (0.022 mmol) of intermediate C5 with 12.7 mg (0.024 mmol) of intermediate L8 in the presence of 9.9 mg (0.026 mmol) of HATU and 19 μl of N , N -diisopropylethylamine. The mixture was stirred at room temperature for 30 minutes, and the product was purified by preparative HPLC and then lyophilized from acetonitrile/water.

The resulting intermediate was taken up in 3 ml of dichloromethane and deblocked with 1 ml of trifluoroacetic acid. After stirring at room temperature for 30 minutes, the preparation was concentrated and the residue was lyophilized from acetonitrile/water. This gave 8.2 mg (36% of theory over 2 steps) of the title compound.

HPLC (Method 11): R _t = 1.8 min;

LC-MS (Method 1): R _t = 0.87 min; MS (ESIpos): m/z = 913 (M+H) ⁺ .

Intermediate F35

N-[31-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatriacontane-1-yl]-L-valyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-N5-carbamoyl-L-ornithinamide

Under argon and at 0°C, 57.3 mg (0.07 mmol) of N-[31-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatriacontane-1-yl]-L-valyl-N ⁵ 1-Carbamoyl-L-ornithine (Intermediate L38), 9.2 mg (0.07 mmol) of HOAt, and 32 mg (0.08 mmol) of HATU were added to 31.8 mg (0.07 mmol) of N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide (Intermediate C40) in 4.0 mL of DMF. 23.5 μL (0.14 mmol) of N , N -diisopropylethylamine were then added and the mixture was stirred at room temperature overnight. 7.7 μL of HOAc was added to the reaction mixture and the mixture was purified directly by preparative RP-HPLC (column: Reprosil 250x30; 10 µm, flow rate: 50 mL/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 33.4 mg (38% of theory) of the title compound.

LC-MS (Method 1): R _t = 1.12 min; MS (ESIpos): m/z = 651 [M+2H] ²⁺ .

Intermediate F36

N-[31-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatriacontane-1-yl]-L-valyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-L-alaninamide

The synthesis of the title compound was carried out analogously to the preparation of intermediate F35.

15.4 mg (0.03 mmol) N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide (Intermediate C40).

25.0 mg (0.03 mmol) N-[31-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatriacontane-1-oyl]-L-valyl-L-alanine (Intermediate L25).

This gave 10.7 mg (27% of theory) of the title compound.

LC-MS (method 1): R _t = 1.13 min; MS (ESIpos): m/z = 1215 [M+H] ⁺ .

Intermediate F37

Trifluoroacetic acid/N-(4-{[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]methyl}pyrrolidin-3-yl)-31-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatriacontane-1-amide (1:1)

mixture of diastereomers

Compound 3-{[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]methyl}-4-{[31-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatriacontane-1-yl]amino}pyrrolidine-1-carboxylic acid tert-butyl ester was prepared similarly to the synthesis of intermediate C21.

8.0 mg (0.01 mmol) and 13.0 mg (0.02 mmol) of tert-butyl 3-amino-4-{[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]methyl}pyrrolidine-1-carboxylate (Intermediate C23).

9.0 mg (0.01 mmol) and 14.7 mg (0.02 mmol) of 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-{27-[(2,5-dioxopyrrolidin-1-yl)oxy]-27-oxo-3,6,9,12,15,18,21,24-octaoxaheptacosan-1-yl}propanamide.

Yield (combining both preparations):

10.5 mg (42%) diastereomer 1

11.6 mg (46%) diastereomer 2

The title compound was prepared analogously to the synthesis of intermediate F38.

10.5 mg (0.01 mmol) tert-butyl 3-{[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]methyl}-4-{[31-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatriacontanoyl]amino}pyrrolidine-1-carboxylate (diastereomer 1)

60.6 mg (0.54 mmol) TFA.

This gave 7.4 mg (70% of theory) of the title compound.

LC-MS (method 1): R _t = 1.09 min; MS (ESIpos): m/z = 1086 [M+H] ⁺ .

Intermediate F38

Trifluoroacetic acid/N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-7,35-dioxo-10,13,16,19,22,25,28,31-octaoxa-3-thia-6,34-diazaheptatriacontane-1-amide (1:1)

24.8 mg (0.02 mmol) of tert-butyl [38-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,31,37-trioxo-7,10,13,16,19,22,25,28-octaoxa-35-thia-4,32,38-triazatetatecontan-41-yl]carbamate (Intermediate C21) was initially charged in 1.0 ml of dichloromethane and 85.8 mg (0.75 mmol) of TFA was added. The mixture was stirred at room temperature for 16 hours. The solvent was evaporated under vacuum, and the residue was purified directly by preparative RP-HPLC (column: Reprosil 250x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 23.0 mg (95% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.96 min; MS (ESIpos): m/z = 1104 [M+H] ⁺ .

Intermediate F39

N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecanoyl]-L-valyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-L-alaninamide

The title compound was prepared analogously to the synthesis of intermediate F35.

56.1 mg (0.10 mmol) N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecan-1-oyl]-L-valyl-L-alanine (intermediate L44).

45.0 mg (0.10 mmol) N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide (Intermediate C40).

This gave 20.9 mg (21% of theory) of the title compound.

LC-MS (Method 1): R _t = 1.16 min; MS (ESIpos): m/z = 1040 [M+H] ⁺ .

Intermediate F40

Trifluoroacetic acid/N-[(4-{[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]methyl}pyrrolidin-3-yl)methyl]-31-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatriacontane-1-amide (1:1)

Tert-butyl 3-{[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]methyl}-4-[33-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,31-dioxo-6,9,12,15,18,21,24,27-octaoxa-2,30-diazatricaria-1-yl]pyrrolidine-1-carboxylate was prepared analogously to the synthesis of intermediate C21.

25.0 mg (0.04 mmol) trifluoroacetic acid/tert-butyl 3-(aminomethyl)-4-{[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]methyl}pyrrolidine-1-carboxylate (1:1) (Intermediate C24).

27.6 mg (0.04 mmol) 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-{27-[(2,5-dioxopyrrolidin-1-yl)oxy]-27-oxo-3,6,9,12,15,18,21,24-octaoxaheptacosan-1-yl}propanamide.

Yield: 20.6 mg (39% of theory)

LC-MS (Method 1): R _t = 1.23 min; MS (ESIpos): m/z = 1200 [M+H] ⁺ .

The title compound was prepared analogously to the synthesis of intermediate F37.

26.1 mg (0.02 mmol) of tert-butyl 3-{[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]methyl}-4-[33-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,31-dioxo-6,9,12,15,18,21,24,27-octaoxa-2,30-diazatricaria-1-yl]pyrrolidine-1-carboxylate.

90.6 mg (0.80 mmol) TFA.

This gave 22.9 mg (95% of theory) of the title compound.

LC-MS (method 1): R _t = 0.91 and 0.92 min; MS (ESIpos): m/z = 1100 [M+H] ⁺ .

Intermediate F41

Trifluoroacetic acid/N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-7,35-dioxo-10,13,16,19,22,25,28,31-octaoxa-3-thia-6,34-diazaheptatriacontane-1-amide 3-oxide (1:1)

The title compound was prepared analogously to intermediate F38 from 15.5 mg (0.01 mmol) of intermediate C22. This gave 4.0 mg (27% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.90 min; MS (ESIpos): m/z = 1120 [M+H] ⁺ .

Intermediate F42

Trifluoroacetic acid/4-{[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]methyl}-N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]pyrrolidine-3-carboxamide (1:1)

40.5 mg (0.06 mmol) of 4-{[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]methyl}-1-(tert-butoxycarbonyl)pyrrolidine-3-carboxylic acid (Intermediate C25) and 14.5 mg (0.08 mmol) of 1-(2-aminoethyl)-1H-pyrrole-2,5-dione hydrochloride (1:1) were initially charged in 1.0 ml of acetonitrile, 64.4 mg (0.51 mmol) of N , N -diisopropylethylamine and 50.0 mg (0.08 mmol) of T3P were added and stirred at room temperature for 16 hours. The same amount of 1-(2-aminoethyl)-1H-pyrrole-2,5-dione hydrochloride (1:1), N , N -diisopropylethylamine, and T3P were added and stirred at room temperature for an additional 4 hours. The reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 125x30; 10µ, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 7.2 mg (15% of theory) of the compound tert-butyl 3-{[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]methyl}-4-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]carbamoyl}pyrrolidine-1-carboxylate.

LC-MS (Method 1): R _t = 1.30 min; MS (ESIpos): m/z = 763 [M+H] ⁺ .

7.2 mg (0.01 mmol) of tert-butyl 3-{[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]methyl}-4-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]carbamoyl}pyrrolidine-1-carboxylate was initially charged in 1.0 mL of dichloromethane, and 43.0 mg (0.38 mmol) of TFA was added. The reaction mixture was stirred at room temperature for 16 hours. The solvent was evaporated under vacuum, and the residue was purified by preparative RP-HPLC (column: Reprosil 125x30; 10µm, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 4.5 mg (50% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.92 min; MS (ESIpos): m/z = 663 [M+H] ⁺ .

Intermediate F43

N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecanoyl]-L-valyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-N5-carbamoyl-L-ornithinamide

22.4 mg (0.03 mmol) of N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecan-1-yl]-L-valyl-N5-carbamoyl-L-ornithine (Intermediate L42) were dissolved in 2.0 ml of DMF and 4.5 mg (0.03 mmol) of HOAt, 15.8 mg (0.04 mmol) of HATU and 15.7 mg (0.03 mmol) of N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide (Intermediate C40) were added. 8.6 mg (0.07 mmol) of N , N -diisopropylethylamine were added, and the reaction mixture was stirred at room temperature overnight. The reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 250x30; 10µ, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 16.7 mg (45% of theory) of the title compound.

LC-MS (Method 4): R _t = 1.34 min; MS (ESIpos): m/z = 1125 [M+H] ⁺ .

Intermediate F44

Trifluoroacetic acid/N-[(4-{[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]methyl}pyrrolidin-3-yl)methyl]-19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecan-1-amide (1:1)

The title compound was prepared analogously to the synthesis of intermediate F40.

25.0 mg (0.04 mmol) trifluoroacetic acid/tert-butyl 3-(aminomethyl)-4-{[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]methyl}pyrrolidine-1-carboxylate (1:1) (Intermediate C24).

20.5 mg (0.04 mmol) 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-{15-[(2,5-dioxopyrrolidin-1-yl)oxy]-15-oxo-3,6,9,12-tetraoxopentadec-1-yl}propanamide.

This gave 21.8 mg (48% of theory) of the compound tert-butyl 3-{[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]methyl}-4-[21-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,19-dioxo-6,9,12,15-tetraoxa-2,18-diazaheneicosan-1-yl]pyrrolidine-1-carboxylate.

LC-MS (Method 1): R _t = 1.22 min; MS (ESIpos): m/z = 1025 [M+H] ⁺ .

21.0 mg (0.02 mmol) tert-butyl 3-{[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]methyl}-4-[21-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,19-dioxo-6,9,12,15-tetraoxa-2,18-diazaheneicostan-1-yl]pyrrolidine-1-carboxylate.

168.3 mg (1.48 mmol) TFA.

This gave 17.3 mg (90% of theory) of the title compound.

LC-MS (method 1): R _t = 0.92 and 0.94 min; MS (ESIpos): m/z = 924 [M+H] ⁺ .

Intermediate F45

Trifluoroacetic acid/N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecanoyl]-L-valyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-L-lysinamide (1:1)

The synthesis was performed similarly to that of intermediate F46.

22.9 mg (0.05 mmol) N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide (Intermediate C40).

36.2 mg (0.05 mmol) N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecan-1-oyl]-L-valyl-N ⁶ -(tert-butoxycarbonyl)-L-lysine (intermediate L41).

This gave 19.8 mg (34%) of the compound N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecan-1-oyl]-L-valyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-N ⁶ -(tert-butoxycarbonyl)-L-lysinamide.

LC-MS (Method 1): R _t = 1.20 min; MS (ESIpos): m/z = 1196 [M+H] ⁺ .

17.0 mg (0.01 mmol) of N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecan-1-yl]-L-valyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-N ⁶ -(tert-butoxycarbonyl)-L-lysinamide.

This gave 13.6 mg (79% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.94 min; MS (ESIpos): m/z = 1096 [M+H] ⁺ .

Intermediate F46

Trifluoroacetic acid/N-[31-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatriacontane-1-yl]-L-valyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-L-lysinamide (1:1)

Under argon and at 0°C, 49.0 mg (0.05 mmol) of N-[31-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatriacontane-1-yl]-L-valyl-N6-(tert-butoxycarbonyl)-L-lysine (intermediate L40), 7.3 1 mg (0.05 mmol) of HOAt and 25.3 mg (0.07 mmol) of HATU were added to 25.1 mg (0.05 mmol) of N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide (Intermediate C40) in 2.0 mL of DMF. 18.6 μL (0.11 mmol) of N , N -diisopropylethylamine were then added, and stirring was continued at room temperature overnight. The reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 250x30; 10 µm, flow rate: 50 mL/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 29.2 mg (37% of theory) of the compound N-[31-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatriacontane-1-yl]-L-valyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-N ⁶ -(tert-butoxycarbonyl)-L-lysinamide.

LC-MS (Method 4): R _t = 1.51 min; MS (ESIpos): m/z = 1372 [M+H] ⁺ .

25.2 mg (0.02 mmol) of N-[31-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatriacontane-1-yl]-L-valyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-N6-(tert-butoxycarbonyl)-L-lysinamide was initially charged in 3.0 ml of dichloromethane, and 83.7 mg (0.73 mmol) of TFA was added. The reaction solution was stirred at room temperature for 48 hours. The solvent was evaporated under vacuum, and the residue was purified by preparative RP-HPLC (column: Reprosil 250x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 24.5 mg (96% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.95 min; MS (ESIpos): m/z = 1272 [M+H] ⁺ .

Intermediate F47

N-[67-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-65-oxo-4,7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61-icosaoxa-64-azahexaheptadecanoyl]-L-valyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-N5-carbamoyl-L-ornithinamide

15.2 mg (0.01 mmol) of N-[67-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-65-oxo-4,7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61-icosaoxa-64-azahexadecanoyl]-L-valyl-N5-carbamoyl-L-ornithine The acid (Intermediate L43) was dissolved in 1.0 ml of DMF, and 1.5 mg (0.01 mmol) of HOAt, 5.2 mg (0.01 mmol) of HATU, and 5.2 mg (0.01 mmol) of N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide (Intermediate C40) were added. 2.9 mg (0.02 mmol) of N , N -diisopropylethylamine was added, and the reaction mixture was stirred at room temperature overnight. The reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 250x30; 10 µm, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 6.8 mg (33% of theory) of the title compound.

LC-MS (Method 4): R _t = 1.37 min; MS (ESIpos): m/z = 1831 [M+H] ⁺ .

Intermediate F48

Trifluoroacetic acid/N1-(3-aminopropyl)-N1-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-N18-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]-6,9,12,15-tetraoxa-3-thiaoctadecane-1,18-diamide (1:1)

Initially, 16.1 mg (0.02 mmol) of tert-butyl [22-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4,21-dioxo-7,10,13,16-tetraoxa-19-thia-3,22-diazapentacosan-25-yl]carbamate (Intermediate C18) was added to 1.5 mL of dichloromethane and 26 μL (0.34 mmol) of TFA was added. The mixture was stirred at room temperature overnight, and then an additional 26 μL (0.34 mmol) of TFA was added. The mixture was stirred again at room temperature overnight. The solvent was evaporated under vacuum, and the residue was purified by preparative RP-HPLC (column: Reprosil 250x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was taken up in water and lyophilized. This yielded 10.8 mg (66% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.98 min; MS (ESIpos): m/z = 857 [M+H] ⁺ .

Intermediate F49

(25S)-25-Amino-22-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4,21-dioxo-7,10,13,16-tetraoxa-19-thia-3,22-diazahexacosanoic acid/trifluoroacetic acid (1:1)

The synthesis of compound (2S)-tert-butyl 4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(chloroacetyl)amino]-2-[(tert-butoxycarbonyl)amino]butanoate was carried out similarly to the synthesis of intermediate C16.

50.0 mg (0.08 mmol) intermediate C2

20.3 mg (0.18 mmol) chloroacetyl chloride

19.0 mg (0.19 mmol) triethylamine

This gave 43.1 mg (77% of theory) of the compound tert-butyl (2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(chloroacetyl)amino]-2-[(tert-butoxycarbonyl)amino]butanoate.

LC-MS (Method 1): R _t = 1.55 min; MS (ESIpos): m/z = 689 [M+H] ⁺ .

The synthesis of compound (6S)-9-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-6-(tert-butoxycarbonyl)-2,2-dimethyl-4,10-dioxo-3,15,18,21,24-pentaoxa-12-thia-5,9-diazaheptacosane-27-oic acid was carried out similarly to the synthesis of intermediate C17.

38.8 mg (0.06 mmol) of tert-butyl (2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(chloroacetyl)amino]-2-[(tert-butoxycarbonyl)amino]butanoate.

19.1 mg (0.07 mmol) 1-sulfanyl-3,6,9,12-tetraoxapentadecan-15-oic acid

45.9 mg (0.14 mmol) cesium carbonate

This gave 40.7 mg (77% of theory) of the compound (6S)-9-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-6-(tert-butoxycarbonyl)-2,2-dimethyl-4,10-dioxo-3,15,18,21,24-pentaoxa-12-thia-5,9-diazaheptacosacan-27-oic acid.

LC-MS (Method 1): R _t = 1.45 min; MS (ESIpos): m/z = 935 [M+H] ⁺ .

The synthesis of compound (25S)-22-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-25-[(tert-butoxycarbonyl)amino]-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4,21-dioxo-7,10,13,16-tetraoxa-19-thia-3,22-diazahexacosano-26-oic acid tert-butyl ester was carried out similarly to the synthesis of intermediate C18.

37.4 mg (0.04 mmol) (6S)-9-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-6-(tert-butoxycarbonyl)-2,2-dimethyl-4,10-dioxo-3,15,18,21,24-pentaoxa-12-thia-5,9-diazaheptacosane-27-oic acid.

9.2 mg (0.05 mmol) 1-(2-aminoethyl)-1H-pyrrole-2,5-dione hydrochloride (1:1).

This gave 23.4 mg (49% of theory) of the compound (25S)-22-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-25-[(tert-butoxycarbonyl)amino]-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4,21-dioxo-7,10,13,16-tetraoxa-19-thia-3,22-diazahexacosano-26-oic acid tert-butyl ester.

LC-MS (Method 1): R _t = 1.47 min; MS (ESIpos): m/z = 1057 [M+H] ⁺ .

The synthesis of the title compound was carried out analogously to the synthesis of intermediate F38.

20.8 mg (0.02 mmol) of (25S)-tert-butyl 22-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-25-[(tert-butoxycarbonyl)amino]-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4,21-dioxo-7,10,13,16-tetraoxa-19-thia-3,22-diazahexacosanoate.

157.0 mg (1.37 mmol) TFA.

This yielded 13.0 mg (65%) of the title compound.

LC-MS (Method 1): R _t = 0.93 min; MS (ESIpos): m/z = 901 [M+H] ⁺ .

Intermediate F50

Trifluoroacetic acid/1-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)-N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]cyclopropanecarboxamide (1:1)

15 mg (0.024 mmol) of Intermediate C5 was dissolved in 6.5 mL of DCM, and 19 mg (0.049 mmol) of Intermediate L24, 13 μL of N , N -diisopropylethylamine, and 10 mg (0.037 mmol) of 2-bromo-1-ethylpyridinium tetrafluoroborate were added. The mixture was stirred at room temperature for 3 hours and then dried under vacuum. The residue was purified by preparative HPLC to yield 2.4 mg of the protected intermediate.

These were then taken up in 1 ml of DCM and deprotected with 0.1 ml of trifluoroacetic acid. Lyophilization from acetonitrile/water yielded 2.6 mg (11% of theory over 2 steps) of the title compound.

HPLC (Method 11): R _t = 2.4 min.

LC-MS (Method 1): R _t = 1.25 min; MS (ESIpos): m/z = 819 [M+H] ⁺ .

Intermediate F51

3-{3-[(3-amino-2-{[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]methyl}propyl)sulfanyl]-2,5-dioxopyrrolidin-1-yl}-N-[17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-15-oxo-4,7,10-trioxa-14-azaheptadecan-1-yl]propanamide (Isomer 1)

10.0 mg (18.079 μmol) of N-[3-amino-2-(sulfanylmethyl)propyl]-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide hydrochloride (1:1) (Isomer 1) was initially loaded in 100 μl of PBS buffer (Sigma D8537) and 200 μl of ACN. 17.1 mg (32.543 μmol) of 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-[17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-15-oxo-4,7,10-trioxa-14-azaheptadecan-1-yl]propanamide was added and stirred at room temperature for 16 hours. The reaction solution was purified by preparative HPLC (eluent: ACN/water + 0.1% TFA, gradient) and lyophilized. This gave 14.0 mg (75% of theory) of the target compound.

Isomer 1

LC-MS (Method 1): R _t = 0.94 min; MS (ESIpos): m/z = 1039 [M+H] ⁺ .

Intermediate F52

3-{3-[(3-amino-2-{[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]methyl}propyl)sulfanyl]-2,5-dioxopyrrolidin-1-yl}-N-[17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-15-oxo-4,7,10-trioxa-14-azaheptadecan-1-yl]propanamide (isomer 2)

6.5 mg (10.694 μmol, LC/MS purity = 91%) of N-[3-amino-2-(sulfanylmethyl)propyl]-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide hydrochloride (1:1) (isomer 2) were initially loaded in 100 μl of PBS buffer (Sigma D8537) and 200 μl of ACN. 10.1 mg (19.249 μmol) of 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-[17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-15-oxo-4,7,10-trioxa-14-azaheptadecan-1-yl]propanamide were added and stirred at room temperature for 16 hours. The reaction solution was purified by preparative HPLC (eluent: ACN/water + 0.1% TFA, gradient) and lyophilized. This yielded 5.0 mg (45% of theory) of the target compound.

Isomer 2

LC-MS (Method 5): R _t = 2.87 min; MS (ESIpos): m/z = 1039 [M+H] ⁺ .

Intermediate F53

N-{3-amino-2-[({1-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl]-2,5-dioxopyrrolidin-3-yl}sulfanyl)methyl]propyl}-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide (Isomer 1)

Initially, 10.0 mg (18.079 μmol) of N-[3-amino-2-(sulfanylmethyl)propyl]-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide hydrochloride (1:1) (Isomer 1) was added to 200 μl of PBS buffer (Sigma D8537) and 400 μl of ACN. 8.1 mg (32.543 μmol) of 1,1'-butane-1,4-diylbis(1H-pyrrole-2,5-dione) was added and stirred at room temperature for 1 hour. Then, 300 μl of DMF was added and stirred for an additional 1.5 hours. The reaction solution was purified by preparative HPLC (eluent: ACN/water + 0.1% TFA, gradient) and lyophilized. This gave 4.0 mg (29% of theory) of the target compound.

Isomer 1

LC-MS (Method 1): R _t = 0.97 min; MS (ESIpos): m/z = 765 [M+H] ⁺ .

Intermediate F54

N-{3-amino-2-[({1-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl]-2,5-dioxopyrrolidin-3-yl}sulfanyl)methyl]propyl}-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide (Isomer 2)

Initially, 5.0 mg (9.040 μmol) of N-[3-amino-2-(sulfanylmethyl)propyl]-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide hydrochloride (1:1) (Isomer 2) was added to 500 μL of DMF. 4.0 mg (16.271 μmol) of 1,1'-butane-1,4-diylbis(1H-pyrrole-2,5-dione) was added and stirred at room temperature for 16 hours. The reaction solution was purified by preparative HPLC (eluent: ACN/water + 0.1% TFA, gradient) and lyophilized. This yielded 1.1 mg (16% of theory) of the target compound.

Isomer 2

LC-MS (Method 6): R _t = 2.41 min; MS (ESIpos): m/z = 765 [M+H] ⁺ .

Intermediate F55

N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecanoyl]-L-valyl-N-{4-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}carbamoyl]phenyl}-L-alaninamide

6.5 mg (4.5 μmol) of N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecanoyl]-L-valyl-N-[4-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}{3-[(tert-butoxycarbonyl)amino]propyl}carbamoyl)phenyl]-L-alaninamide were dissolved in 441 μl of dichloromethane, and 44 μl (573.1 μmol) of trifluoroacetic acid were added. The mixture was concentrated on a rotary evaporator at room temperature, taken up in water and ACN, and lyophilized. This yielded 5.6 mg (94% of theory, purity according to LC/MS = 92%) of the target compound.

LC-MS (Method 1): R _t = 1.01 min; MS (ESIpos): m/z = 1100.6 [M+H] ⁺ .

Intermediate F56

Trifluoroacetic acid/(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-N-[(2S)-1-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino}-1-oxopropan-2-yl]butanamide (1:1)

The title compound was prepared analogously to intermediate F50.

LC-MS (Method 1): R _t = 0.9 min; MS (EIpos): m/z = 708 [M+H] ⁺ .

Intermediate F57

Trifluoroacetic acid/(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-N-[(2R)-1-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino}-1-oxopropan-2-yl]butanamide (1:1)

The title compound was prepared analogously to intermediate F56.

LC-MS (Method 1): R _t = 0.91 min; MS (EIpos): m/z = 708 [M+H] ⁺ .

Intermediate F58

N-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}-L-valyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-L-alaninamide

16.2 mg (0.02 mmol) of trifluoroacetic acid/L-valyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-L-alaninamide (1:1) (Intermediate C41) was initially charged in 1.0 ml of DMF, and 8.3 mg (0.06 mmol) of N , N -diisopropylethylamine and 12.6 mg (0.04 mmol) of 1,1'-[(1,5-dioxopentan-1,5-diyl)bis(oxy)]dipyrrolidine-2,5-dione were added. The reaction mixture was stirred at room temperature overnight and directly purified by preparative RP-HPLC (column: Reprosil 250x30; 10µm, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum and the residue was dried under high vacuum. This gave 11.0 mg (60% of theory) of the title compound.

LC-MS (Method 1): R _t = 1.18 min; MS (ESIpos): m/z = 852 [M+H] ⁺ .

Intermediate F82

N-[31-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatriacontane-1-yl]-L-valyl-N-{3-[{1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-L-alaninamide

The synthesis of the title compound was carried out analogously to the synthesis of intermediate F83. The racemic intermediates used were obtained analogously to the corresponding R-isomer intermediates.

LC-MS (Method 2): R _t = 7.07 min; MS (EIpos): m/z = 1236 [M+Na] ⁺ .

Intermediate F83

N-[31-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatriacontane-1-yl]-L-valyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-L-alaninamide

30.0 mg (0.06 mmol) of N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide (Example 98) and 26.1 mg (0.06 mmol) of N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-alanine 2,5-dioxopyrrolidin-1-yl ester were initially charged in 2.0 ml of DMF, and 19.4 mg (0.19 mmol) of 4-methylmorpholine were added. The reaction mixture was stirred at room temperature overnight, and 11.5 mg (0.19 mmol) of HOAc was added. The reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 250x30; 10µm, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 41.9 mg (79% of theory) of the compound 9H-fluoren-9-ylmethyl [(2S)-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}amino)-1-oxopropan-2-yl]carbamate.

LC-MS (Method 1): R _t = 1.44 min; MS (ESIpos): m/z = 763 [M+H] ⁺ .

37.2 mg (0.05 mmol) of 9H-fluoren-9-ylmethyl [(2S)-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}amino)-1-oxopropan-2-yl]carbamate were dissolved in 1.5 ml of DMF, and 124.6 mg (1.46 mmol) of 2-aminoethanol were added. The reaction mixture was stirred overnight at room temperature. The reaction mixture was partitioned between ethyl acetate and water, and the organic phase was washed twice with water and once with saturated NaCl solution. After drying over magnesium sulfate, the solvent was evaporated under vacuum, and the residue was purified on silica gel (eluent: dichloromethane/methanol 10:1). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 14.2 mg (50% of theory) of the compound N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-L-alaninamide.

LC-MS (method 1): R _t = 0.92 min; MS (ESIpos): m/z = 541 [M+H] ⁺ .

14.1 mg (0.03 mmol) of N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-L-alaninamide and 11.4 mg (0.03 mmol) of N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-valine 2,5-dioxopyrrolidin-1-yl ester were dissolved in 1.5 ml of DMF, and 7.9 mg (0.08 mmol) of 4-methylmorpholine was added. The reaction mixture was stirred at room temperature overnight, and 4.7 mg (0.08 mmol) of HOAc was added. The reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 250x30; 10µm, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 15.9 mg (71% of theory) of the compound N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-valyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-L-alaninamide.

LC-MS (Method 1): R _t = 1.46 min; MS (ESIpos): m/z = 862 (M+H) ⁺ .

14.9 mg (0.02 mmol) of N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-valyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-L-alaninamide was dissolved in 1.5 ml of DMF, and 44.2 mg (0.52 mmol) of 2-aminoethanol was added. The reaction mixture was stirred at room temperature overnight. The reaction mixture was partitioned between ethyl acetate and water, and the organic phase was washed twice with water and once with saturated NaCl solution. After drying over magnesium sulfate, the solvent was evaporated under vacuum, and the residue was purified on silica gel (eluent: dichloromethane/methanol 10:1). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 5.7 mg (52% of theory) of the compound L-valyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-L-alaninamide.

LC-MS (Method 1): R _t = 0.92 min; MS (ESIpos): m/z = 640 (M+H) ⁺ .

5.5 mg (8.6 μmol) of L-valyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-L-alaninamide and 6.5 mg (6.5 μmol) of 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-{27-[(2,5-dioxopyrrolidin-1-yl)oxy]-27-oxo-3,6,9,12,15,18,21,24-octaoxaheptacosan-1-yl}propanamide were dissolved in 1.0 ml of DMF, and 0.9 mg (8.6 mmol) of 4-methylmorpholine was added. The reaction mixture was stirred at room temperature overnight, and 0.8 mg (0.01 mmol) of HOAc was added. The reaction mixture was purified directly by preparative RP-HPLC (column: Reprosil 250x30; 10µ, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 7.7 mg (74% of theory) of the title compound.

LC-MS (Method 1): R _t = 1.10 min; MS (ESIpos): m/z = 1214 (M+H) ⁺ .

Intermediate F84

Trifluoroacetic acid/(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-N-(3-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino}-3-oxopropyl)butanamide (1:1)

First, 16.5 mg (0.02 mmol) of intermediate C54 was placed in 5 ml of DMF and reacted with 10.4 mg (0.041 mmol) of trifluoroacetic acid/1-(2-aminoethyl)-1H-pyrrole-2,5-dione (1:1) in the presence of 11.7 mg (0.03 mmol) of HATU and 18 μl of N , N -diisopropylethylamine. After stirring at room temperature for 5 minutes, the mixture was concentrated and the residue was taken up in acetonitrile/water (1:1). The pH was adjusted to 2 with trifluoroacetic acid and the preparation was reconcentrated. The remaining residue was purified by preparative HPLC. This yielded 8 mg (42% of theory) of the protected intermediate.

LC-MS (Method 1): R _t = 1.38 min; MS (EIpos): m/z = 929 [M+H] ⁺ .

7.6 mg (0.008 mmol) of this intermediate were placed in 3 ml of DMF and 92 mg (0.82 mmol) of 1,4-diazabicyclo[2.2.2]octane were added. The preparation was treated in an ultrasonic bath for 1 hour. 31 μl of acetic acid were then added and the preparation was concentrated under high vacuum. The residue was purified by preparative HPLC. This yielded 3 mg (45% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.86 min; MS (EIpos): m/z = 707 [M+H] ⁺ .

¹ H NMR (500 MHz, DMSO-d ₆ ): d = 8.15 (t, 1H), 7.9-8.1 (m, 4H), 7.6 (m,1H), 7.5 (s, 1H), 7.15-7.35 (m, 6H), 6.9-7.0 (m, 3H), 6.85 (s, 1H), 5.6 (s,1H), 4.9 and 5.2 (2d, 2H), 4.05 and 4.2 (2d, 2H), 3.1-3.2 (m, 4H), 2.15 (m, 2H), 0.7 and 1.45 (2m, 2H), 0.8 (s, 9H).

Intermediate F85

Trifluoroacetic acid/(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]butanamide (1:1)

First, 10 mg (0.014 mmol) of intermediate C53 was placed in 3.4 mL of DMF and reacted with 7 mg (0.027 mmol) of trifluoroacetic acid/1-(2-aminoethyl)-1H-pyrrole-2,5-dione (1:1) in the presence of 7.8 mg (0.02 mmol) of HATU and 12 μL of N , N -diisopropylethylamine. After stirring at room temperature for 15 minutes, the mixture was concentrated, and the residue was purified by preparative HPLC. This yielded 6.6 mg (57% of theory) of the protected intermediate.

LC-MS (Method 1): R _t = 1.4 min; MS (EIpos): m/z = 858 [M+H] ⁺ .

6.6 mg (0.008 mmol) of this intermediate were placed in 2 ml of DMF and 86 mg (0.77 mmol) of 1,4-diazabicyclo[2.2.2]octane were added. The preparation was treated in an ultrasonic bath for 2 hours. 44 μl of acetic acid were then added and the preparation was concentrated under high vacuum. The residue was purified by preparative HPLC. This yielded 3.3 mg (53% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.88 min; MS (EIpos): m/z = 636 [M+H] ⁺ .

Intermediate F86

Trifluoroacetic acid/(2S)-2-amino-4-[{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-N-(3-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino}-3-oxopropyl)butanamide (1:1)

The title compound was prepared from 8 mg (0.012 mmol) of intermediate C51 by reaction with 4.5 mg (0.017 mmol) of trifluoroacetic acid/1-(2-aminoethyl)-1H- pyrrole -2,5-dione (1:1) in the presence of 5.8 mg (0.015 mmol) of HATU and 10 μl of N ,N-diisopropylethylamine, followed by deprotection with trifluoroacetic acid. This yielded 7 mg (78% of theory over 2 steps).

LC-MS (Method 1): R _t = 0.83 min; MS (EIpos): m/z = 708 [M+H] ⁺ .

Intermediate F87

Trifluoroacetic acid/(2S)-2-amino-4-[{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-N-(3-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino}-3-oxopropyl)butanamide (1:1)

The title compound was prepared analogously to Intermediate F2 from 16 mg (0.025 mmol) of Intermediate C49 by reaction with 24 mg (0.076 mmol) of Intermediate L1 in the presence of EDCI/HOBT and N , N -diisopropylethylamine, followed by deprotection with trifluoroacetic acid. This yielded 3 mg of the title compound (14% of theory over 2 steps).

LC-MS (Method 1): R _t = 0.88 min; MS (EIpos): m/z = 694 [M+H] ⁺ .

Intermediate F88

This compound was prepared analogously to intermediate F8.

LC-MS (Method 5): R _t = 2.97 min; MS (EIpos): m/z = 1006 [M+H] ⁺ .

Intermediate F89

Trifluoroacetic acid/N-{(2S)-2-amino-4-[{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl-L-alanyl-N ⁵ -carbamoyl-N-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl]-L-ornithinamide (1:1)

The title compound was prepared from 8 mg (0.012 mmol) of intermediate C51 by reaction with 7.4 mg (0.014 mmol) of intermediate L8 in the presence of 5.8 mg (0.015 mmol) of HATU and 10 μl of N , N -diisopropylethylamine, followed by deprotection with trifluoroacetic acid. This yielded 10 mg (78% of theory over 2 steps).

LC-MS (Method 1): R _t = 0.87 min; MS (EIpos): m/z = 984 [M+H] ⁺ .

Intermediate F90

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N ⁵ -carbamoyl-L-ornithine-N ⁶ -{(2S)-2-amino-4-[{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butyryl}-L-lysine/trifluoroacetic acid (1:1)

The title compound was prepared from 11 mg (0.018 mmol) of intermediate C49 by reaction with 13.7 mg (0.018 mmol) of intermediate L17 in the presence of 34 mg (0.089 mmol) of HATU and 19 μl of N , N -diisopropylethylamine, followed by deprotection with trifluoroacetic acid. This yielded 7.5 mg (35% of theory over 2 steps).

LC-MS (Method 8): R _t = 6.78 min; MS (EIpos): m/z = 1092 [M+H] ⁺ .

Intermediate F91

Trifluoroacetic acid/(2S)-2-amino-4-[{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]butanamide (1:1)

Dissolve 9.3 mg (0.01 mmol) of tert-butyl [(2S)-4-[{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-1-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino}-1-oxobutan-2-yl]carbamate in 2 ml of dichloromethane, add 740 mg (6.49 mmol, 0.50 ml) of trifluoroacetic acid, and stir at room temperature for 1.5 hours. The reaction mixture is then concentrated, and the residue is taken up in acetonitrile and water and lyophilized. This yields 9.2 mg (96% of theory) of the target compound.

LC-MS (Method 1): R _t = 0.88 min; MS (EIpos): m/z = 637 [M+H] ⁺ .

Intermediate F103

N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecan-1-yl]-L-valyl-N-(3-{[(1R)-1-(3-benzyl-7-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)-2-methylpropyl](4-methylbenzoyl)amino}propyl)-L-alaninamide

The title compound was prepared from 10 mg (0.019 mmol) of N-(3-aminopropyl)-N-[(1R)-1-(3-benzyl-7-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)-2-methylpropyl]-4-methylbenzamide by reaction with 11.3 mg (0.019 mmol) of intermediate L44 in the presence of 8.8 mg (0.023 mmol) of HATU and 10 μL of N , N -diisopropylethylamine. Purification was by preparative HPLC.

Yield: 8.5 mg (35% of theory)

LC-MS (Method 5): R _t = 3.82 min; MS (EIpos): m/z = 1085 [M+H] ⁺ .

Intermediate F104

Trifluoroacetic acid/(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-N-(2-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}ethyl)butanamide (1:1)

10 mg (0.014 mmol) of intermediate C53 was dissolved in 3.3 ml of DMF, and 8.5 mg (0.027 mmol) of intermediate L1, 7.8 mg (0.02 mmol) of HATU, and 12 μl of N , N -diisopropylethylamine were added. The preparation was stirred at room temperature for 15 minutes and then concentrated. The residue was purified by preparative HPLC, yielding 5.6 mg (38% of theory) of the protected intermediate after lyophilization.

LC-MS (Method 1): R _t = 1.32 min; MS (ESIpos): m/z = 915 (M+H) ⁺ .

5.6 mg (0.006 mmol) of this intermediate were placed in 2 ml of DMF and 69 mg (0.61 mmol) of 1,4-diazabicyclo[2.2.2]octane were added. The preparation was treated in an ultrasonic bath for 2 hours. 35 μl of acetic acid were then added and the preparation was concentrated under high vacuum. The residue was purified by preparative HPLC. This yielded 2.4 mg (48% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.84 min; MS (EIpos): m/z = 693 [M+H] ⁺ .

HPLC (Method 11): R _t = 1.91 min.

Alternatively, the title compound can also be prepared from intermediate C58. Initially, 15 mg (0.023 mmol) of intermediate C58 was reacted with 11 mg (0.036 mmol) of intermediate L1 in the presence of 13 mg (0.034 mmol) of HATU and 10 μL of N , N -diisopropylethylamine. After stirring at room temperature for 60 minutes, the mixture was concentrated, and the residue was purified by preparative HPLC. This yielded 12.3 mg (63% of theory) of the protected intermediate.

LC-MS (Method 1): R _t = 1.3 min; MS (EIpos): m/z = 837 [M+H] ⁺ .

In the second step, this intermediate was dissolved in 3 ml of 2,2,2-trifluoroethanol. 12 mg (0.088 mmol) of zinc chloride was added and the mixture was stirred at 50°C for 2 hours. Then, 26 mg (0.088 mmol) of ethylenediamine-N,N,N',N'-tetraacetic acid and 2 ml of 0.1% aqueous trifluoroacetic acid were added. The preparation was purified by preparative HPLC. Concentration of the appropriate fractions and lyophilization of the residue from acetonitrile/water yielded 8.1 mg (68% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.89 min; MS (ESIpos): m/z = 693 (M+H) ⁺ .

Intermediate F105

N ² -{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]-L-glutamine/trifluoroacetic acid (1:1)

The title compound was prepared in analogy to intermediate F32 from intermediate C5 and intermediate L46.

LC-MS (Method 1): R _t = 0.82 min; MS (EIpos): m/z = 766 [M+H] ⁺ .

Intermediate F106

Trifluoroacetic acid/N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl-L-alanyl-N5-carbamoyl-N-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl]-L-ornithinamide (1:1)

The title compound was prepared in analogy to intermediate F104 from intermediate C53 and intermediate L47.

HPLC (Method 11): R _t = 1.85 min;

LC-MS (Method 1): R _t = 0.86 min; MS (ESIpos): m/z = 983 (M+H) ⁺ .

Intermediate F107

Trifluoroacetic acid/(1R,2S)-2-({(2S)-2-amino-4-[{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)-N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]cyclopentanecarboxamide (1:1)

The title compound was prepared from 15 mg (0.024 mmol) of intermediate C49 by reaction with 22.3 mg (0.049 mmol) of intermediate L48 in the presence of 14 mg (0.037 mmol) of HATU and 21 μl of N , N -diisopropylethylamine, followed by deprotection with trifluoroacetic acid. This yielded 13 mg (60% of theory over 2 steps).

LC-MS (Method 1): R _t = 0.9 min; MS (EIpos): m/z = 748 [M+H] ⁺ .

Intermediate F108

Trifluoroacetic acid/(1R,2S)-2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)-N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]cyclopentanecarboxamide (1:1)

The title compound was prepared analogously to intermediate F104 from 20 mg (0.027 mmol) of intermediate C53 and 24 mg (0.054 mmol) of intermediate L48. This gave 3 mg (14% of theory over 2 steps).

LC-MS (Method 1): R _t = 0.93 min; MS (EIpos): m/z = 747 [M+H] ⁺ .

Intermediate F109

Trifluoroacetic acid/(2S)-2-amino-4-[{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-N-{2-[(bromoacetyl)amino]ethyl}butanamide (1:1)

17 mg (0.026 mmol) of intermediate C57 was dissolved in 3 mL of DMF and reacted with 7 mg (0.027 mmol) of commercially available 1-(2-bromoacetoxy)pyrrolidine-2,5-dione in the presence of 14 μL of N , N -diisopropylethylamine. After stirring at room temperature for 15 minutes, the mixture was concentrated, and the residue was purified by preparative HPLC. This yielded 7 mg (33% of theory) of this intermediate.

LC-MS (method 1): R _t = 1.29 min; MS (ESIpos): m/z = 777 and 779 (M+H) ⁺ .

This intermediate was taken up in 1 ml of dichloromethane and deprotected with 1 ml of trifluoroacetic acid. After concentration and lyophilization from acetonitrile/water, 6 mg (88% of theory) of the title compound were obtained.

LC-MS (Method 1): R _t = 0.86 min; MS (ESIpos): m/z = 677/679 (M+H) ⁺ .

Intermediate F110

N-(Bromoacetyl)-L-valyl-L-alanyl-N6-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butyryl}-L-lysine/trifluoroacetic acid (1:1)

The title compound was prepared analogously to intermediate F109 from 16 mg (0.023 mmol) of intermediate C5 and 17 mg (0.025 mmol) of intermediate L49. This gave 6 mg (24% of theory over 2 steps) of the title compound.

HPLC (Method 11): R _t = 1.93 min;

LC-MS (method 1): R _t = 0.88 min; MS (ESIpos): m/z = 933 and 935 (M+H) ⁺ .

Intermediate F111

Trifluoroacetic acid/(1S,3R)-3-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)-N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]cyclopentanecarboxamide (1:1)

The title compound was prepared from 15 mg (0.022 mmol) of intermediate C5 by reaction with 16 mg (0.044 mmol) of intermediate L50 in the presence of 12.5 mg (0.032 mmol) of HATU and 19 μl of N , N -diisopropylethylamine, followed by deprotection with trifluoroacetic acid. This yielded 13 mg (67% of theory over 2 steps).

LC-MS (Method 1): R _t = 0.89 min; MS (EIpos): m/z = 748 [M+H] ⁺ .

Intermediate F112

Trifluoroacetic acid/(1S,3R)-3-({(2S)-2-amino-4-[{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)-N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]cyclopentanecarboxamide (1:1)

The title compound was prepared from 15 mg (0.024 mmol) of intermediate C49 by reaction with 18 mg (0.049 mmol) of intermediate L50 in the presence of 14 mg (0.037 mmol) of HATU and 21 μl of N , N -diisopropylethylamine, followed by deprotection with trifluoroacetic acid. This yielded 12 mg (51% of theory over 2 steps).

LC-MS (Method 1): R _t = 0.89 min; MS (EIpos): m/z = 748 [M+H] ⁺ .

Intermediate F113

Trifluoroacetic acid/(1S,3R)-3-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)-N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]cyclopentanecarboxamide (1:1)

The title compound was prepared from 15 mg (0.019 mmol) of intermediate C53 by reaction with 14 mg (0.038 mmol) of intermediate L50 in the presence of 11 mg (0.029 mmol) of HATU and 17 μl of N , N -diisopropylethylamine, followed by deprotection with 133 mg of DABCO in 2 ml of DMF. Purification by HPLC yielded 4 mg (24% of theory over 2 steps).

LC-MS (Method 5): R _t = 2.77 min; MS (EIpos): m/z = 747 [M+H] ⁺ .

Intermediate F114

Trifluoroacetic acid/(1R,3R)-3-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)-N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]cyclopentanecarboxamide (1:1)

The title compound was prepared from 15 mg (0.022 mmol) of intermediate C5 by reaction with 16 mg (0.044 mmol) of intermediate L51 in the presence of 12.6 mg (0.032 mmol) of HATU and 19 μl of N , N -diisopropylethylamine, followed by deprotection with trifluoroacetic acid. This yielded 11 mg (53% of theory over 2 steps).

LC-MS (Method 1): R _t = 0.89 min; MS (EIpos): m/z = 748 [M+H] ⁺ .

Intermediate F115

Trifluoroacetic acid/(1R,3R)-3-({(2S)-2-amino-4-[{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)-N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]cyclopentanecarboxamide (1:1)

The title compound was prepared from 15 mg (0.024 mmol) of intermediate C49 by reaction with 18 mg (0.047 mmol) of intermediate L51 in the presence of 13 mg (0.035 mmol) of HATU and 21 μl of N , N -diisopropylethylamine, followed by deprotection with trifluoroacetic acid. This yielded 12 mg (51% of theory over 2 steps).

LC-MS (Method 1): R _t = 0.87 min; MS (EIpos): m/z = 748 [M+H] ⁺ .

Intermediate F116

Trifluoroacetic acid/(1R,3R)-3-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)-N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]cyclopentanecarboxamide (1:1)

The title compound was prepared from 11 mg (0.014 mmol) of intermediate C51 by reaction with 11 mg (0.028 mmol) of intermediate L51 in the presence of 8 mg (0.021 mmol) of HATU and 12 μl of N , N -diisopropylethylamine, followed by deprotection with 87 mg of DABCO in 2 ml of DMF. Purification by HPLC yielded 3.3 mg (28% of theory over 2 steps).

LC-MS (Method 1): R _t = 0.92 min; MS (EIpos): m/z = 747 [M+H] ⁺ .

Intermediate F117

Trifluoroacetic acid/N-[(3S)-3-amino-4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxobutyl]-N-{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide (1:1)

The title compound was prepared from intermediate C49 according to classical peptide chemistry methods. First, C49 was coupled with 9H-fluoren-9-ylmethyl hydrazinecarboxylate in the presence of HATU. The Fmoc protecting group was then removed with piperidine in DMF, and the resulting hydrazide was coupled with 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid in the presence of HATU. In the final step, the Boc protecting group was removed with TFA in dichloromethane.

LC-MS (Method 1): R _t = 0.93 min; MS (EIpos): m/z = 722 [M+H] ⁺ .

Intermediate F118

Trifluoroacetic acid/N-[(3S)-3-amino-4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxobutyl]-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide (1:1)

In the first step, the title compound was prepared analogously to Intermediate F3 from 15 mg (0.019 mmol) of Intermediate C53 by coupling with commercially available 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexane hydrazide in the presence of HATU. The Fmoc protecting group was then cleaved using 142 mg of DABCO in DMF. Purification by HPLC yielded 3 mg (19% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.90 min; MS (EIpos): m/z = 721 [M+H] ⁺ .

Intermediate F119

Trifluoroacetic acid/(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-N-{2-[(bromoacetyl)amino]ethyl}butanamide (1:1)

29 mg (0.044 mmol) of intermediate C58 were placed in 3.4 ml of DMF, and 36 mg (0.087 mmol) of intermediate L52, 25 mg (0.065 mmol) of HATU, and 19 μl of N , N -diisopropylethylamine were added. After stirring at room temperature for 60 minutes, the mixture was concentrated, and the residue was purified by preparative HPLC. This yielded 26.4 mg (73% of theory) of the intermediate.

LC-MS (method 1): R _t = 1.34 min; MS (ESIpos): m/z = 820 and 822 (M+H) ⁺ .

This intermediate was dissolved in 3 ml of 2,2,2-trifluoroethanol. 6.5 mg (0.048 mmol) of zinc chloride was added, and the preparation was stirred at 50°C for 4 hours. 13.9 mg (0.048 mmol) of ethylenediamine-N,N,N',N'-tetraacetic acid and 2 ml of 0.1% aqueous trifluoroacetic acid were added. The preparation was purified by preparative HPLC. Concentration of the appropriate fractions and lyophilization of the residue from acetonitrile/water yielded 14.4 mg (58% of theory) of the title compound.

LC-MS (method 1): R _t = 0.88 min; MS (ESIpos): m/z = 676 and 678 (M+H) ⁺ .

Intermediate F120

Trifluoroacetic acid/(1S,3S)-3-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)-N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]cyclopentanecarboxamide (1:1)

The title compound was prepared from 10 mg (0.015 mmol) of intermediate C5 by reaction with 11 mg (0.03 mmol) of intermediate L53 in the presence of 8.4 mg (0.022 mmol) of HATU and 13 μl of N , N -diisopropylethylamine, followed by deprotection with trifluoroacetic acid. This yielded 7.5 mg (59% of theory over 2 steps).

LC-MS (Method 1): R _t = 0.85 min; MS (EIpos): m/z = 748 [M+H] ⁺ .

Intermediate F121

Trifluoroacetic acid/(1S,3S)-3-({(2S)-2-amino-4-[{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)-N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]cyclopentanecarboxamide (1:1)

The title compound was prepared from 10 mg (0.016 mmol) of intermediate C49 by reaction with 11.5 mg (0.031 mmol) of intermediate L53 in the presence of 9 mg (0.024 mmol) of HATU and 14 μl of N , N -diisopropylethylamine, followed by deprotection with trifluoroacetic acid. This yielded 9 mg (61% of theory over 2 steps).

LC-MS (Method 1): R _t = 0.84 min; MS (EIpos): m/z = 748 [M+H] ⁺ .

Intermediate F122

Trifluoroacetic acid/(1S,3S)-3-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)-N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]cyclopentanecarboxamide (1:1)

The title compound was prepared from 15 mg (0.019 mmol) of intermediate C53 by reaction with 14 mg (0.038 mmol) of intermediate L53 in the presence of 11 mg (0.029 mmol) of HATU and 17 μl of N , N -diisopropylethylamine, followed by deprotection with 202 mg of DABCO in 3 ml of DMF. Purification by HPLC yielded 4 mg (24% of theory over 2 steps).

LC-MS (Method 1): R _t = 0.87 min; MS (EIpos): m/z = 747 [M+H] ⁺ .

Intermediate F123

Trifluoroacetic acid/(1R,3S)-3-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)-N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]cyclopentanecarboxamide (1:1)

The title compound was prepared from 10 mg (0.015 mmol) of intermediate C5 by reaction with 11 mg (0.030 mmol) of intermediate L54 in the presence of 8.4 mg (0.022 mmol) of HATU and 13 μl of N , N -diisopropylethylamine, followed by deprotection with trifluoroacetic acid. This yielded 4 mg (31% of theory over 2 steps).

LC-MS (Method 1): R _t = 0.86 min; MS (EIpos): m/z = 748 [M+H] ⁺ .

Intermediate F124

Trifluoroacetic acid/(1R,3S)-3-({(2S)-2-amino-4-[{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)-N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]cyclopentanecarboxamide (1:1)

The title compound was prepared from 10 mg (0.016 mmol) of intermediate C49 by reaction with 11.5 mg (0.031 mmol) of intermediate L54 in the presence of 9 mg (0.024 mmol) of HATU and 14 μl of N , N -diisopropylethylamine, followed by deprotection with trifluoroacetic acid. This yielded 9 mg (66% of theory over 2 steps).

LC-MS (Method 1): R _t = 0.84 min; MS (EIpos): m/z = 748 [M+H] ⁺ .

Intermediate F125

Trifluoroacetic acid/(1R,3S)-3-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)-N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]cyclopentanecarboxamide (1:1)

The title compound was prepared from 15 mg (0.019 mmol) of intermediate C53 by reaction with 14 mg (0.038 mmol) of intermediate L54 in the presence of 11 mg (0.029 mmol) of HATU and 17 μl of N , N -diisopropylethylamine, followed by deprotection with 127 mg of DABCO in 3 ml of DMF. Purification by HPLC yielded 3 mg (17% of theory over 2 steps).

LC-MS (Method 4): R _t = 1.08 min; MS (EIpos): m/z = 769 [M+Na] ⁺ .

Intermediate F126

N-(Bromoacetyl)-L-valyl-L-alanyl-N ⁶ -{(2S)-2-amino-4-[{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butyryl}-L-lysine/trifluoroacetic acid (1:1)

The title compound was prepared analogously to intermediate F110 from 18 mg (0.027 mmol) of intermediate C49 and 21 mg (0.027 mmol) of intermediate L49. This gave 8.7 mg (30% of theory over 2 stages) of the title compound.

HPLC (Method 11): R _t = 1.94 min;

LC-MS (method 1): R _t = 0.86 min; MS (ESIpos): m/z = 933 and 935 (M+H) ⁺ .

Intermediate F127

Trifluoroacetic acid/(2S)-2-amino-4-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}[(2S)-2-methoxypropionyl]amino)-N-(2-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}ethyl)butanamide (1:1)

12 mg (0.015 mmol) of Intermediate C59 was dissolved in 2.4 ml of DMF, and 14.6 mg (0.046 mmol) of Intermediate L1, 6 mg (0.031 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 5.9 mg (0.039 mmol) of 1-hydroxy- 1H -benzotriazole hydrate, and 8 μl of N , N -diisopropylethylamine were added. After stirring at room temperature for 1 hour, the mixture was concentrated, and the residue was purified by preparative HPLC. This yielded 11 mg (70% of theory) of this intermediate.

LC-MS (Method 1): R _t = 1.34 min; MS (ESIpos): m/z = 942 (M+H) ⁺ .

11 mg (0.011 mmol) of this intermediate were placed in 2 ml of DMF and 123 mg (1.1 mmol) of 1,4-diazabicyclo[2.2.2]octane were added. The preparation was treated in an ultrasonic bath for 2 hours. 63 μl of acetic acid were then added and the preparation was concentrated under high vacuum. The residue was purified by preparative HPLC. This yielded 2 mg (22% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.89 min; MS (EIpos): m/z = 721 [M+H] ⁺ .

HPLC (Method 11): _Rt = 1.95 min.

Intermediate F128

N ⁶ -(N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-D-alanyl)-N ² -{N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-L-alanyl}-L-lysine/trifluoroacetic acid (1:1)

The title compound was prepared from 3 mg (0.005 mmol) of intermediate C5 by reaction with 2.5 mg (0.007 mmol) of intermediate L55 in the presence of 2.5 mg (0.003 mmol) of HATU and 3 μl of N , N -diisopropylethylamine, followed by deprotection with trifluoroacetic acid. This yielded 1.4 mg (32% of theory over 2 steps).

LC-MS (Method 1): R _t = 0.93 min; MS (EIpos): m/z = 1077 [M+H] ⁺ .

Intermediate F129

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-L-alanyl-N ⁶ -{[(1R,3S)-3-({(2S)-2-amino-4-[{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butanoyl}amino)cyclopentyl]carbonyl}-L-lysine/trifluoroacetic acid

The title compound was prepared analogously to Intermediate F128 from 10 mg (0.016 mmol) of Intermediate C49 by reaction with 19 mg (0.024 mmol) of Intermediate L56 in the presence of 12 mg (0.031 mmol) of HATU and 14 μl of N , N -diisopropylethylamine, followed by deprotection with trifluoroacetic acid. This yielded 13.5 mg (70% of theory over 2 steps).

LC-MS (Method 1): R _t = 0.9 min; MS (EIpos): m/z = 1117 [M+H] ⁺ .

Intermediate F142

R/S-{2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}-N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecanoyl]-homocysteine/trifluoroacetic acid (1:1)

20.0 mg (23.7 μmol) of R/S-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl)-homocysteine/trifluoroacetic acid (1:1) and 13 0.4 mg (26.04 mmol) of 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-{15-[(2,5-dioxopyrrolidin-1-yl)oxy]-15-oxo-3,6,9,12-tetraoxapentadecan-1-yl}propanamide was dissolved in 1.0 mL of DMF, and 4.8 mg (47.34 μmol) of 4-methylmorpholine was added. The reaction mixture was stirred at room temperature overnight. 3.6 mg (0.06 mmol) of acetic acid was added, and the reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 250x30; 10 µm, flow rate: 50 mL/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 12.4 mg (44% of theory) of the compound R/S-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl)-N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecan-1-oyl]homocysteine.

LC-MS (Method 1): R _t = 1.30 min; MS (ESIpos): m/z = 1129 (M+H) ⁺ .

10.0 mg (8.85 μmol) of R/S-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl)-N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecan-1-oyl]-L-homocysteine was dissolved in trifluoroethanol, and 3.1 mg (22.71 μmol) of zinc dichloride was added. The reaction mixture was stirred at 50°C overnight. To the reaction mixture was added 3.9 mg (0.01 mmol) of ethylenediamine-N,N,N',N'-tetraacetic acid, stirred briefly, and then water (0.1% TFA) was added. Purification was carried out directly by preparative RP-HPLC (column: Reprosil 250x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was lyophilized with a small amount of water. This yielded 7.6 mg (78% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.94 min; MS (ESIpos): m/z = 983 (M+H) ⁺ .

¹ H NMR (400 MHz, DMSO-d ₆ ): δ [ppm] = 0.50 (m, 1H), 0.81 (s, 9H), 1.49(m, 1H), 1.89 (m, 1H), 2.05 (m, 1H), 2.29-2.43 (m, 4H), 2.45-2.55 (m, 2H),2.58-2.74 (m, 2H), 3.10-3.20 (m, 2H), 3.21-3.40 (m, 2H), 3.42-3.54 (m, 16H),3.55-3.65 (m, 4H), 4.28 (m, 1H), 4.91 (dd, 1H), 5.18 (dd, 1H), 5.60 (s, 1H), 6.95 (m, 1H), 7.00 (s, 2H), 7.15-7.38 (m, 7H), 7.53 (s, 1H), 7.68 (m, 1H), 8.00 (m, 2H).

Intermediate F143

Trifluoroacetic acid/6-({2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}sulfanyl)-N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]hexanamide (1:1)

30.0 mg (0.05 mmol) of 2-(trimethylsilyl)ethyl {3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(chloroacetyl)amino]propyl}carbamate and 13.5 mg (0.07 mmol) of 6-(acetylsulfanyl)hexanoic acid were initially added together in 2.0 mL of methanol containing one drop of water. 23.0 mg (0.17 mmol) of potassium carbonate were added. The reaction mixture was stirred at 50°C for 4 hours. Ethyl acetate was added to the reaction mixture. The organic phase was washed with saturated NaCl solution and dried over magnesium sulfate. The solvent was evaporated under vacuum. The residue was purified by preparative RP-HPLC (column: Reprosil 125x30; 10µm, flow rate: 50 mL/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 54.2 mg (90% of theory) of the compound 11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-14-thia-7,11-diaza-2-silaicosanoic acid.

LC-MS (Method 1): R _t = 1.49 min; MS (ESIpos): m/z = 1106 (M+H) ⁺ .

54.0 mg (0.07 mmol) of 11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-14-thia-7,11-diaza-2-silaicosane-20-oic acid and 16.7 mg (0.09 mmol) of 1-(2-aminoethyl)-1H-pyrrole-2,5-dione hydrochloride (1:1) were initially charged in 3.0 ml of acetonitrile, and 75.0 mg (0.58 mmol) of N , N -diisopropylethylamine was added. 60.0 mg (0.09 mmol) of T3P (50% in acetonitrile) was added, and the mixture was stirred at room temperature overnight. The reaction was quenched with water, and the reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 125x30; 10µm, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 42.8 mg (68% of theory) of the compound [2-(trimethylsilyl)ethyl 3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}{[(6-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino}-6-oxohexyl)sulfanyl]acetyl}amino)propyl]carbamate.

LC-MS (Method 1): R _t = 1.48 min; MS (ESIpos): m/z = 866 (M+H) ⁺ .

20.0 mg (0.02 mmol) of 2-(trimethylsilyl)ethyl [3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}{[(6-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino}-6-oxohexyl)sulfanyl]acetyl}amino)propyl]carbamate was dissolved in 2.0 ml of trifluoroethanol, and 4.7 mg (0.04 mmol) of zinc dichloride was added. The reaction mixture was stirred at 50°C overnight, and then 10.1 mg (0.04 mmol) of ethylenediamine-N,N,N',N'-tetraacetic acid was added, followed by stirring for 10 minutes. Water (0.1% TFA) was added, and the reaction mixture was purified by preparative RP-HPLC (column: Reprosil 125x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 9.2 mg (48% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.98 min; MS (ESIpos): m/z = 722 (M+H) ⁺ .

Intermediate F144

Trifluoroacetic acid/N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide (1:1)

50.0 mg (0.1 mmol) of tert-butyl [3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino)propyl]carbamate (Intermediate C15) were initially charged in 2.0 ml of dichloromethane and 22.7 mg (0.22 mmol) of triethylamine and 49.3 mg (0.22 mmol) of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl chloride (Intermediate L60) WISV1648-1-1 were added.

The reaction mixture was stirred at room temperature overnight. Then, 1 equivalent of intermediate L60 and 1.2 equivalents of triethylamine were added three times every 2 hours, followed by stirring at room temperature overnight. This procedure was repeated twice more. The solvent was removed under vacuum, and the residue was purified by preparative RP-HPLC (column: Reprosil 125x30; 10µ, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 30.9 mg (43% of theory) of the compound [tert-butyl 3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino)propyl]carbamate.

LC-MS (Method 1): R _t = 1.51 min; MS (ESIpos): m/z = 706 (M+H) ⁺ .

24.6 mg (0.04 mmol) of tert-butyl [3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino)propyl]carbamate was dissolved in 3.0 ml of dichloromethane, 79.5 mg (0.7 mmol) of TFA was added, and the mixture was stirred at room temperature for 6 hours. An additional 79.5 mg (0.7 mmol) of TFA was added, and the mixture was stirred at room temperature overnight. The solvent was removed under vacuum, and the residue was co-distilled three times with dichloromethane. The residue was purified by preparative RP-HPLC (column: Reprosil 125x30; 10µm, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum and the residue was dried under high vacuum. This gave 24.2 mg (97% of theory) of the title compound.

LC-MS (Method 1): R _t = 1.02 min; MS (ESIpos): m/z = 606 (M+H) ⁺ .

Intermediate F145

Trifluoroacetic acid/6-({2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}sulfanyl)-N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]hexanamide

90.0 mg (0.15 mmol) of tert-butyl {3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(chloroacetyl)amino]propyl}carbamate (Intermediate C16) and 43.6 mg (0.23 mmol) of 6-(acetylsulfanyl)hexanoic acid were initially charged in 9.0 ml of methanol containing one drop of water, and 73.9 mg (0.54 mmol) of potassium carbonate was added. The reaction mixture was stirred at room temperature for 4 hours, after which ethyl acetate was added. The organic phase was washed with water/saturated NaCl solution and then with saturated NaCl solution. The organic phase was dried over magnesium sulfate, and the solvent was evaporated under vacuum. The residue was purified on silica gel (eluent: dichloromethane/methanol 100:2). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 98.7 mg (83% of theory) of the compound 9-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-4,10-dioxo-3-oxa-12-thia-5,9-diazaoctadecane-18-oic acid.

LC-MS (Method 1): R _t = 1.44 min; MS (ESIpos): m/z = 701 (M+H) ⁺ .

20.0 mg (0.03 mmol) of 9-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-4,10-dioxo-3-oxa-12-thia-5,9-diazaoctadecane-18-oic acid and 6.5 mg (0.04 mmol) of 1-(2-aminoethyl)-1H-pyrrole-2,5-dione hydrochloride (1:1) were initially charged in 1.5 ml of acetonitrile, and 23.6 mg (0.04 mmol) of T3P and 29.5 mg (0.23 mmol) of N , N -diisopropylethylamine were added. The reaction mixture was stirred at room temperature overnight, and then water was added. The reaction mixture was purified by preparative RP-HPLC (column: Reprosil 250x30; 10µ, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 16.7 mg (99% of theory) of the compound tert-butyl [3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}{[(6-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino}-6-oxohexyl)sulfanyl]acetyl}amino)propyl]carbamate.

LC-MS (Method 1): R _t = 1.40 min; MS (ESIpos): m/z = 823 (M+H) ⁺ .

14.8 mg (0.02 mmol) of tert-butyl [3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}{[(6-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino}-6-oxohexyl)sulfanyl]acetyl}amino)propyl]carbamate was dissolved in 1.5 ml of dichloromethane, and 41.0 mg (0.36 mmol) of TFA was added. The reaction mixture was stirred at room temperature overnight.

Then, two additional 41.0 mg (0.36 mmol) of TFA were added each time and stirred at room temperature overnight. The solvent was evaporated under vacuum, and the residue was purified by preparative RP-HPLC (column: Reprosil 250x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was taken up in 1,4-dioxane and water and lyophilized. This yielded 2.9 mg (19% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.93 min; MS (ESIpos): m/z = 723 (M+H) ⁺ .

Intermediate F146

R/S-[2-([(3S)-3-amino-3-carboxypropyl]{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)-2-oxoethyl]-N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecanoyl]homocysteine/trifluoroacetic acid (1:1)

25.0 mg (28.12 μmol) of R/S-[(8S)-11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-8-carboxy-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl]homocysteine (Intermediate C12) was added. The product was dissolved in 2.0 mL of DMF and 15.9 mg (30.93 μmol) of 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-{15-[(2,5-dioxopyrrolidin-1-yl)oxy]-15-oxo-3,6,9,12-tetraoxapentadecan-1-yl}propanamide. 11.4 mg (112.48 μmol) of 4-methylmorpholine were added. The reaction mixture was stirred at room temperature overnight. 7.6 mg (0.13 mmol) of acetic acid was added, and the reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 250x30; 10 µm, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 23.9 mg (59% of theory) of the compound R/S-[(8S)-11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-8-carboxy-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl]-N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecan-1-oyl]homocysteine.

LC-MS (Method 1): R _t = 1.26 min; MS (ESIpos): m/z = 1173 (M+H) ⁺ .

11.8 mg (8.23 μmol) of R/S-[(8S)-11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-8-carboxy-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl]-N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecan-1-oyl]homocysteine was dissolved in trifluoroethanol, and 1.7 mg (12.35 μmol) of zinc dichloride was added. The reaction mixture was stirred at 50°C overnight. To the reaction mixture was added 3.6 mg (0.01 mmol) of ethylenediamine-N,N,N',N'-tetraacetic acid, stirred briefly, and then water (0.1% TFA) was added. Purification was carried out directly by preparative RP-HPLC (column: Reprosil 250x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 5.8 mg (62% of theory) of the title compound.

LC-MS (Method 4): R _t = 1.20 min; MS (ESIpos): m/z = 1029 (M+H) ⁺ .

Intermediate F147

Trifluoroacetic acid/N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-10-oxo-3,6-dioxa-16-thia-9-azaoctadecane-18-carboxamide (1:1)

Initially, 15.0 mg (0.03 mmol) of 9-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-4,10-dioxo-3-oxa-12-thia-5,9-diazaoctadecane-18-oic acid (Intermediate C13) was charged in 1.5 mL of acetonitrile and 22.1 mg (0.17 mmol) of N , N -diisopropylethylamine was added, followed by 17.7 mg (0.03 mmol) of T3P. The mixture was stirred at room temperature for 5 minutes, followed by the addition of 9.5 mg (0.03 mmol) of trifluoroacetic acid/1-{2-[2-(2-aminoethoxy)ethoxy]ethyl}-1H-pyrrole-2,5-dione (1:1) (Intermediate L59). The reaction mixture was stirred at room temperature overnight and quenched with water. The reaction mixture was purified directly by preparative RP-HPLC (column: Reprosil 250x30; 10µ, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 14.8 mg (57% of theory) of tert-butyl [19-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-10,18-dioxo-3,6-dioxa-16-thia-9,19-diazadocosan-22-yl]carbamate.

LC-MS (Method 1): R _t = 1.40 min; MS (ESIpos): m/z = 911 (M+H) ⁺ .

14.2 mg (0.02 mmol) of tert-butyl [19-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-10,18-dioxo-3,6-dioxa-16-thia-9,19-diazadocosan-22-yl]carbamate was dissolved in 1.5 ml of dichloromethane, 35.5 mg (0.31 mmol) of TFA was added, and the mixture was stirred at room temperature overnight. An additional 71.0 mg (0.62 mmol) of TFA was added, and the mixture was stirred at room temperature overnight. The solvent was evaporated under vacuum, and the residue was purified by preparative RP-HPLC (column: Reprosil 250x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, the residue was taken up in a small amount of water and lyophilized. This yielded 14.0 mg (97% of theory) of the title compound.

LC-MS (Method 1): R _t = 1.01 min; MS (ESIpos): m/z = 811 (M+H) ⁺ .

Intermediate F148

Trifluoroacetic acid/6-({2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}sulfinyl)-N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]hexanamide (1:1)

The title compound was formed as a by-product in the synthesis of intermediate F145. This gave 8.1 mg (53% of theory) of the title compound.

LC-MS (Method 1): R _t = 1.25 min; MS (ESIpos): m/z = 739 [M+H] ⁺ .

Intermediate F149

Trifluoroacetic acid/R/S-{2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}-N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecanoyl]homocysteine (1:1)

20.0 mg (24.94 μmol) of R/S-[2-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}{3-[(tert-butoxycarbonyl)amino]propyl}amino)-2-oxoethyl]homocysteine (Intermediate C14) and 14.1 mg (27.44 μmol) of 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-{15-[(2,5-dioxopyrrolidin-1-yl)oxy]-15-oxo-3,6,9,12-tetraoxapentadecan-1-yl}propanamide were initially charged in 1.0 ml of DMF and 5.1 mg (49.88 μmol) of 4-methylmorpholine were added. The reaction mixture was stirred overnight. 3.7 mg (0.06 mmol) of acetic acid were added, and the reaction mixture was purified directly by preparative RP-HPLC (column: Reprosil 250x30; 10µm, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 18.2 mg (67% of theory) of the compound R/S-[2-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}{3-[(tert-butoxycarbonyl)amino]propyl}amino)-2-oxoethyl]-N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azadecanoyl]homocysteine.

LC-MS (Method 1): R _t = 1.23 min; MS (ESIpos): m/z = 1086 (M+H) ⁺ .

17.6 mg (0.02 mmol) of R/S-[2-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}{3-[(tert-butoxycarbonyl)amino]propyl}amino)-2-oxoethyl]-N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecanoyl]homocysteine was dissolved in 1.5 ml of dichloromethane, 37.0 mg (0.32 mmol) of TFA was added, and the mixture was stirred at room temperature overnight. An additional 74.0 mg (0.64 mmol) of TFA was added, and the mixture was stirred at room temperature overnight. The solvent was evaporated under vacuum, and the residue was purified by preparative RP-HPLC (column: Reprosil 250x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, the residue was taken up in a small amount of water and lyophilized. This yielded 16.0 mg (90% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.92 min; MS (ESIpos): m/z = 986 (M+H) ⁺ .

Intermediate F150

Trifluoroacetic acid/N-[31-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatriacontanoyl]-L-valyl-N-[2-({2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}sulfanyl)ethyl]-L-alaninamide (1:1)

Under argon at 0°C, 10.0 mg (0.02 mmol) of trifluoroacetic acid/tert-butyl [3-({[(2-aminoethyl)sulfanyl]acetyl}{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino)propyl]carbamate (Intermediate C20) was reacted in 1.0 ml of DMF with 12.1 mg (0.02 mmol) of tert-butyl trifluoroacetate. The product was treated with 2.2 mg (0.02 mmol) of N-[31-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatriacontane-1-yl]-L-valyl-L-alanine (Intermediate L25), 2.2 mg (0.02 mmol) of HOAt, and 7.6 mg (0.02 mmol) of HATU. 5.5 μL (0.03 mmol) of N , N -diisopropylethylamine was then added and the mixture was stirred at room temperature overnight. 1.8 μL of HOAc was added to the reaction mixture and the product was purified directly by preparative RP-HPLC (column: Reprosil 250x30; 10 µm, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 10.4 mg (48% of theory) of the compound N-[31-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatriacontane-1-yl]-L-valyl-N-(9-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-4,10-dioxo-3-oxa-12-thia-5,9-diazatetradec-14-yl)-L-alaninamide.

LC-MS (Method 4): R _t = 1.60 min; MS (ESIpos): m/z = 687.5 [M+2H] ²⁺ .

9.5 mg (0.01 mmol) of N-[31-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatriacontane-1-yl]-L-valyl-N-(9-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-4,10-dioxo-3-oxa-12-thia-5,9-diazatetradec-14-yl)-L-alaninamide were initially charged in 1.0 ml of dichloromethane, 15.8 mg (0.14 mmol) of TFA were added and stirred overnight. An additional 31.6 mg (0.28 mmol) of TFA was added, and the mixture was stirred overnight. The solvent was evaporated under vacuum, and the residue was purified by preparative RP-HPLC (column: Reprosil 250x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was taken up in a small amount of water and lyophilized. This yielded 10.2 mg (98% of theory) of the title compound.

LC-MS (Method 4): R _t = 1.13 min; MS (ESIpos): m/z = 637.5 [M+2H] ²⁺ .

Intermediate F151

N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecanoyl]-L-valyl-N-(3-{(2S)-5-(2,5-difluorophenyl)-3-[methoxy(methyl)carbamoyl]-2-phenyl-2,3-dihydro-1,3,4-thiadiazol-2-yl}propyl)-L-alaninamide

5.0 mg (0.01 mmol) of (2S)-2-(3-aminopropyl)-5-(2,5-difluorophenyl)-N-methoxy-N-methyl-2-phenyl-1,3,4-thiadiazole-3(2H)-carboxamide was initially charged in 1.0 mL of acetonitrile, and 7.7 mg (0.06 mmol) of N , N -diisopropylethylamine and 9.8 g (0.02 mmol) of T3P were added. The mixture was stirred at room temperature for 5 minutes, followed by the addition of 9.1 mg (0.02 mmol) of N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecanoyl]-L-valyl-L-alanine (Intermediate L44). The mixture was stirred at room temperature overnight. Water was added, and the reaction mixture was purified directly by preparative RP-HPLC (column: Reprosil 250x40; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 4.3 mg (35% of theory) of the title compound.

LC-MS (Method 1): R _t = 1.02 min; MS (ESIpos): m/z = 989 [M+H] ⁺ .

Intermediate F152

N-[31-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatriacontane-1-yl]-L-valyl-N-(3-{(2S)-5-(2,5-difluorophenyl)-3-[methoxy(methyl)carbamoyl]-2-phenyl-2,3-dihydro-1,3,4-thiadiazol-2-yl}propyl)-L-alaninamide

5.0 mg (0.01 mmol) of (2S)-2-(3-aminopropyl)-5-(2,5-difluorophenyl)-N-methoxy-N-methyl-2-phenyl-1,3,4-thiadiazole-3(2H)-carboxamide was initially charged in 1.0 mL of acetonitrile, and 7.7 mg (0.06 mmol) of N , N -diisopropylethylamine and 9.8 g (0.02 mmol) of T3P were added. The mixture was stirred at room temperature for 5 minutes, and then 11.8 mg (0.02 mmol) of N-[31-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatriacontane-1-yl]-L-valyl-L-alanine (Intermediate L25) was added. The reaction mixture was stirred at room temperature overnight. Water was added and the reaction mixture was purified directly by preparative RP-HPLC (column: Reprosil 250x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 4.7 mg (34% of theory) of the title compound.

LC-MS (Method 4): R _t = 1.34 min; MS (ESIpos): m/z = 1165 [M+H] ⁺ .

Intermediate F153

Trifluoroacetic acid/(2S)-2-amino-4-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}[(2S)-2-hydroxypropionyl]amino)-N-(2-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}ethyl)butanamide (1:1)

The synthesis was carried out analogously to intermediate F104 from intermediate C60.

LC-MS (Method 1): R _t = 1.1 min; MS (ESIpos): m/z = 707 (M+H) ⁺ .

Intermediate F154

N ⁶ -(N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl)-N ² -{N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-L-alanyl}-L-lysine/trifluoroacetic acid (1:1)

The title compound was prepared analogously to intermediate F2 from 10 mg (0.015 mmol) of intermediate C8 and 15 mg (0.022 mmol) of intermediate L6.

HPLC (Method 11): R _t = 1.91 min;

LC-MS (Method 1): R _t = 0.89 min; MS (ESIpos): m/z = 1077 (M+H) ⁺ .

Intermediate F155

N ⁶ -(N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl)-N ² -{N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-L-alanyl}-L-lysine/trifluoroacetic acid (1:1)

The title compound was prepared by coupling 14 mg (0.019 mmol) of intermediate C61 with 15 mg (0.021 mmol) of intermediate L61 in the presence of 8.7 mg (0.023 mmol) of HATU and 17 μl of N , N -diisopropylethylamine and subsequent deprotection with zinc chloride in trifluoroethanol as described for intermediate F119. Purification by preparative HPLC yielded 13 mg (59% of theory over 2 steps) of the title compound.

LC-MS (Method 1): R _t = 0.86 min; MS (ESIpos): m/z = 1076 (M+H) ⁺ .

Intermediate F156

N-(Bromoacetyl)-L-valyl-L-alanyl-N ⁶ -{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butyryl}-L-lysine/trifluoroacetic acid (1:1)

First, the tripeptide derivative L-valyl-L-alanyl-N ⁶ -(tert-butoxycarbonyl)-L-lysine 2-(trimethylsilyl)ethyl ester was prepared from N ² -[(benzyloxy)carbonyl]-N 6 -(tert-butoxycarbonyl)-L-lysine according to the classical methods of peptide chemistry (esterification with 2-(trimethylsilyl)ethanol using EDCI/DMAP, hydrogenolysis, coupling with ^N -[(benzyloxy)carbonyl]-L-valyl-L-alanine in the presence of HATU, and further hydrogenolysis).

84 mg (0.163 mmol) of this intermediate was placed in 2.5 ml of DMF, and 58 mg (0.244 mmol) of 1-(2-bromoacetoxy)pyrrolidine-2,5-dione was added. After stirring at room temperature for 10 minutes, the mixture was concentrated. The residue was taken up in acetonitrile/water (1:1) and adjusted to pH 2 with trifluoroacetic acid. The mixture was then purified by preparative HPLC. After concentration of the appropriate fractions, the residue was taken up in 15 ml of 5% trifluoroacetic acid in DCM and stirred at room temperature for 2 hours. The mixture was then concentrated with slight cooling, and the residue was lyophilized from acetonitrile/water (1:1). 53 mg (50% of theory) of this intermediate was obtained over two steps.

LC-MS (method 1): R _t = 0.72 min; MS (ESIpos): m/z = 537 and 539 (M+H) ⁺ .

To synthesize the title compound, 18 mg (0.027 mmol) of this intermediate was placed in 4 mL of DMF, and 16 mg (0.025 mmol) of intermediate C61, 19 mg of HATU, and 9 μL of N , N -diisopropylethylamine were added. After stirring at room temperature for 5 minutes, a few drops of trifluoroacetic acid were added, and the preparation was purified by preparative HPLC. After concentration of the appropriate fractions and lyophilization from acetonitrile/water (1:1), the resulting intermediate was dissolved in 3 mL of 2,2,2-trifluoroethanol. After the addition of 4.8 mg (0.035 mmol) of zinc chloride, the preparation was stirred at 50°C for 2.5 hours. Then, 10 mg (0.035 mmol) of ethylenediamine-N,N,N',N'-tetraacetic acid was added, and the reaction was diluted with acetonitrile/water and filtered. Purification was then performed by preparative HPLC. Concentration of the appropriate fractions and lyophilization of the residue from acetonitrile/water yielded 3.2 mg (13% of theory) of the title compound over 2 steps.

HPLC (Method 11): R _t = 1.94 min;

LC-MS (Method 5): R _t = 2.79 min; MS (ESIpos): m/z = 932 and 934 (M+H) ⁺ .

Intermediate F163

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-L-alanyl-N6-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butanoyl}-L-lysine/trifluoroacetic acid (1:1)

The title compound was prepared by coupling 37 mg (0.056 mmol) of intermediate C58 and 41 mg (0.056 mmol) of intermediate L61 in the presence of HATU and subsequent deblocking with zinc chloride as described for intermediate F119. This gave 12 mg (19% of theory over 2 steps) of the title compound.

HPLC (Method 11): R _t = 1.49 min;

LC-MS (Method 1): R _t = 0.89 min; MS (ESIpos): m/z = 1005 (M+H) ⁺ .

Intermediate F164

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N5-carbamoyl-L-ornithine-N6-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butyryl}-L-lysine/trifluoroacetic acid (1:1)

The title compound was prepared analogously to intermediate F155 by coupling 20 mg (0.030 mmol) of intermediate C58 with 27 mg (0.033 mmol) of intermediate L62 in the presence of HATU and N , N -diisopropylethylamine and subsequent deprotection with zinc chloride in trifluoroethanol.

HPLC (Method 11): R _t = 1.92 min;

LC-MS (Method 1): R _t = 0.87 min; MS (ESIpos): m/z = 1091 (M+H) ⁺ .

Intermediate F165

N ⁶ -(N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl)-N ² -{N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N 5 -carbamoyl-L-ornithine}-L-lysine/trifluoroacetic acid (1:1)

The title compound was prepared analogously to intermediate F155 by coupling 15 mg (0.021 mmol) of intermediate C61 with 18 mg (0.023 mmol) of intermediate L62 in the presence of HATU and subsequent deprotection with zinc chloride in trifluoroethanol.

LC-MS (Method 1): R _t = 0.88 min; MS (ESIpos): m/z = 1162 (M+H) ⁺ .

Intermediate F166

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N ⁵ -carbamoyl-L-ornithine-N ⁶ -{[(1R,3S)-3-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)cyclopentyl]carbonyl}-L-lysine/trifluoroacetic acid (1:1)

First, trifluoroacetic acid/benzyl (1R, 3S)-3-aminocyclopentanecarboxylate (1:1) was prepared from commercially available (1R, 3S)-3-[(tert-butoxycarbonyl)amino]cyclopentanecarboxylic acid according to the classical methods of peptide chemistry by esterification with benzyl alcohol using EDCI/DMAP and subsequent cleavage of the tert-butoxycarbonyl protecting group with TFA in DCM.

51 mg (0.076 mmol) of this intermediate was placed in 6 ml of DMF and coupled with 50 mg (0.076 mmol) of intermediate C58 in the presence of HATU and N , N -diisopropylethylamine. After purification by preparative HPLC, the intermediate was placed in methanol and hydrogenated over 10% palladium-activated carbon under standard pressure of hydrogen for 2 hours at room temperature. The catalyst was then filtered off, the solvent removed under vacuum, and the product purified by preparative HPLC. Lyophilization from dioxane yielded 21 mg (34% of theory over 2 steps) of (1R,3S)-3-{[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoyl]amino}cyclopentanecarboxylic acid.

The title compound was prepared analogously to intermediate F155 by coupling 10.5 mg (0.013 mmol) of this intermediate with 11.4 mg (0.014 mmol) of intermediate L62 in the presence of HATU and subsequent deprotection with zinc chloride in trifluoroethanol. Purification by preparative HPLC gave 8.6 mg (48% of theory over 2 steps) of the title compound.

LC-MS (Method 1): R _t = 0.88 min; MS (ESIpos): m/z = 1203 (M+H) ⁺ .

Intermediate F167

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-L-alanyl-N ⁶ -{[(1R,3S)-3-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butanoyl}amino)cyclopentyl]carbonyl}-L-lysine/trifluoroacetic acid (1:1)

The title compound was prepared analogously to Intermediate F129 from 11 mg (0.016 mmol) of Intermediate C5 by reaction with 20 mg (0.024 mmol) of Intermediate L56 in the presence of 12 mg (0.032 mmol) of HATU and 14 μl of N , N -diisopropylethylamine, followed by deprotection with trifluoroacetic acid. This yielded 11 mg (46% of theory over 2 steps).

LC-MS (Method 4): R _t = 1.13 min; MS (EIpos): m/z = 1117 [M+H] ⁺ .

Intermediate F168

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-L-alanyl-N ⁶ -{[(1R,2S)-2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)cyclopentyl]carbonyl}-L-lysine/trifluoroacetic acid (1:1)

First, trifluoroacetic acid/benzyl (1R,2S)-2-aminocyclopentanecarboxylate (1:1) was prepared from commercially available (1R,2S)-2-[(tert-butoxycarbonyl)amino]cyclopentanecarboxylic acid according to the classical methods of peptide chemistry by esterification with benzyl alcohol using EDCI/DMAP and subsequent cleavage of the tert-butoxycarbonyl protecting group with TFA in DCM.

102 mg (0.305 mmol) of this intermediate was placed in 12 ml of DMF and coupled with 100 mg (0.152 mmol) of intermediate C58 in the presence of HATU and N , N -diisopropylethylamine. After purification by preparative HPLC, the intermediate was placed in methanol and hydrogenated over 10% palladium-activated carbon under standard pressure of hydrogen for 2 hours at room temperature. The catalyst was then filtered off, the solvent removed under vacuum, and the product purified by preparative HPLC. Lyophilization from acetonitrile/water 1:1 yielded 70 mg (59% of theory over 2 steps) of (1R,2S)-2-{[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoyl]amino}cyclopentanecarboxylic acid.

The title compound was then prepared by coupling 20 mg (0.013 mmol) of this intermediate with 16.6 mg (0.023 mmol) of intermediate L61 in the presence of 9.5 mg (0.025 mmol) of HATU and 18 μl of N , N -diisopropylethylamine and subsequent deprotection with zinc chloride in trifluoroethanol as described for intermediate F119. Purification by preparative HPLC yielded 9.3 mg (30% of theory over 2 steps) of the title compound.

LC-MS (Method 1): R _t = 0.98 min; MS (ESIpos): m/z = 1116 (M+H) ⁺ .

Intermediate F169

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N ⁵ -carbamoyl-L-ornithine-N ⁶ -{[(1R,2S)-2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)cyclopentyl]carbonyl}-L-lysine/trifluoroacetic acid (1:1)

The synthesis of the title compound was carried out analogously to intermediate F168 from intermediates C58 and L62.

LC-MS (Method 1): R _t = 0.91 min; MS (ESIpos): m/z = 1202 (M+H) ⁺ .

Intermediate F170

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N ⁵ -carbamoyl-L-ornithine-N ⁶ -{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butyryl}-D-lysine/trifluoroacetic acid (1:1)

The title compound was prepared analogously to its diastereomeric intermediate F23.

HPLC (Method 11): R _t = 1.9 min;

LC-MS (Method 1): R _t = 0.83 min; MS (ESIpos): m/z = 1092 (M+H) ⁺ .

Intermediate F171

N ⁶ -(N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-D-alanyl)-N ² -{N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-L-alanyl}-L-lysine/trifluoroacetic acid (1:1)

The synthesis of the title compound was carried out analogously to intermediate F155 from intermediates C62 and L61.

LC-MS (Method 1): R _t = 0.93 min; MS (ESIpos): m/z = 1076 (M+H) ⁺ .

Intermediate F172

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-L-alanyl-N-[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]-L-glutamine/trifluoroacetic acid (1:1)

The title compound was prepared from 10 mg (0.013 mmol) of intermediate C63 by coupling with 9 mg (0.014 mmol) of intermediate L63 in the presence of 5.5 mg (0.014 mmol) of HATU and 11 μl of N , N -diisopropylethylamine, followed by deprotection by stirring in a 1:1 trifluoroacetic acid/dichloromethane solution for 2.5 hours. This yielded 11 mg (72% of theory over 2 steps).

LC-MS (Method 1): R _t = 0.9 min; MS (EIpos): m/z = 1049 [M+H] ⁺ .

Intermediate F173

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-L-alanyl-N-[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]-L-glutamine/trifluoroacetic acid (1:1)

The title compound was prepared from 15 mg (0.018 mmol) of intermediate C64 by coupling with 12 mg (0.02 mmol) of intermediate L63 in the presence of 7.7 mg (0.02 mmol) of HATU and 16 μl of N , N -diisopropylethylamine and subsequent deprotection with zinc chloride in trifluoroethanol as described for intermediate F119. Purification by preparative HPLC yielded 12 mg (58% of theory over 2 steps) of the title compound.

LC-MS (Method 1): R _t = 0.91 min; MS (EIpos): m/z = 1048 [M+H] ⁺ .

Intermediate F174

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-L-alanyl-N-[2-({(2S)-2-amino-4-[{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]-L-glutamine/trifluoroacetic acid (1:1)

The title compound was prepared in analogy to intermediate F172 from intermediates C57 and L63.

LC-MS (Method 1): R _t = 0.9 min; MS (EIpos): m/z = 1049 [M+H] ⁺ .

Intermediate F175

Trifluoroacetic acid/N-[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide (1:1)

The title compound was prepared by coupling 11 mg (0.013 mmol) of intermediate C64 with 3.4 mg (0.016 mmol) of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid in the presence of 6.7 mg (0.018 mmol) of HATU and 9 μl of N , N -diisopropylethylamine, and subsequent deprotection with zinc chloride in trifluoroethanol as described for intermediate F119. Purification by preparative HPLC yielded 8 mg (69% of theory over 2 steps) of the title compound.

LC-MS (Method 1): R _t = 1.35 min; MS (EIpos): m/z = 893 [M+H] ⁺ .

Intermediate F176

Trifluoroacetic acid/(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-N-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-12-oxo-3,6,9-trioxa-13-azapentadecan-15-yl]butanamide (1:1)

The title compound was prepared by coupling 5 mg (0.006 mmol) of intermediate C64 with 2 mg (0.007 mmol) of 3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy]ethoxy}ethoxy)propanoic acid (the preparation of which is described under intermediate L15) in the presence of 3.5 mg (0.009 mmol) of HATU and 4 μl of N , N -diisopropylethylamine, and subsequent deprotection with zinc chloride in trifluoroethanol as described for intermediate F119. Purification by preparative HPLC yielded 2 mg (35% of theory over 2 steps) of the title compound.

LC-MS (Method 1): R _t = 0.86 min; MS (EIpos): m/z = 839 [M+H] ⁺ .

Intermediate F177

Trifluoroacetic acid/(1R,2S)-2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)-N-(2-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}ethyl)cyclopentanecarboxamide (1:1)

The title compound was prepared analogously to Intermediate F168 using Intermediate L1 instead of Intermediate L61.

LC-MS (Method 1): R _t = 0.86 min; MS (EIpos): m/z = 804 [M+H] ⁺ .

Intermediate F178

Trifluoroacetic acid/(1R,2S)-2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)-N-{2-[(bromoacetyl)amino]ethyl}cyclopentanecarboxamide (1:1)

The title compound was prepared analogously to Intermediate F177 using Intermediate L52 instead of Intermediate L1.

LC-MS (method 1): R _t = 0.89 min; MS (EIpos): m/z = 787 and 789 [M+H] ⁺ .

Intermediate F179

Trifluoroacetic acid/(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl]butanamide (1:1)

The title compound was prepared by coupling 15 mg (0.023 mmol) of intermediate C58 with 6 mg (0.025 mmol) of 1-(6-aminohexyl)-1H-pyrrole-2,5-dione in the presence of 13 mg (0.034 mmol) of HATU and 16 μl of N , N -diisopropylethylamine, and subsequent deprotection with zinc chloride in trifluoroethanol as described for intermediate F119. Purification by preparative HPLC yielded 8.5 mg (46% of theory over 2 steps) of the title compound.

LC-MS (Method 6): R _t = 2.22 min; MS (EIpos): m/z = 692 [M+H] ⁺ .

Intermediate F180

N-[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]-N2-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-glutamine/trifluoroacetic acid (1:1)

The title compound was prepared by coupling 9.6 mg (0.012 mmol) of intermediate C64 with 5 mg (0.013 mmol) of intermediate L64 in the presence of 7 mg (0.018 mmol) of HATU and 6 μl of N , N -diisopropylethylamine and subsequent deprotection with zinc chloride in trifluoroethanol as described for intermediate F119. Purification by preparative HPLC yielded 3.1 mg (28% of theory over 2 steps) of the title compound.

LC-MS (Method 1): R _t = 0.85 min; MS (EIpos): m/z = 822 [M+H] ⁺ .

Intermediate F192

N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-3-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}-L-alanine/trifluoroacetic acid (1:1)

60 mg (0.091 mmol) of intermediate C58 were placed in 8 ml of DMF and coupled with 45 mg (0.100 mmol) of intermediate L65 in the presence of 42 mg (0.11 mmol) of HATU and 64 microliters of N , N -diisopropylethylamine. After purification by preparative HPLC, the intermediate was placed in 10 ml of ethanol and hydrogenated at room temperature under standard pressure of hydrogen over 10% palladium-activated carbon for 45 minutes. The catalyst was then filtered off, the solvent removed under vacuum, and the product was purified by preparative HPLC. Lyophilization from acetonitrile/water 1:1 yielded 24.5 mg (31% of theory over 2 steps) of 2-(trimethylsilyl)ethyl 3-amino-N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoyl]-L-alaninate.

LC-MS (Method 1): R _t = 1.17 min; MS (EIpos): m/z = 844 [M+H] ⁺ .

The title compound was then prepared by coupling 10 mg (0.012 mmol) of this intermediate with 2 mg (0.013 mmol) of commercially available (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetic acid intermediate in the presence of 5.4 mg (0.014 mmol) of HATU and 8 μl of N , N -diisopropylethylamine and subsequent deprotection with zinc chloride in trifluoroethanol as described for intermediate F119. Purification by preparative HPLC yielded 3.5 mg (33% of theory over 2 steps) of the title compound.

LC-MS (Method 1): R _t = 0.81 min; MS (ESIpos): m/z = 737 (M+H) ⁺ .

Intermediate F193

N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-3-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}-D-alanine/trifluoroacetic acid (1:1)

The synthesis of the title compound was carried out analogously to intermediate F192 from 3-{[(benzyloxy)carbonyl]amino}-N-(tert-butoxycarbonyl)-D-alanine/N-cyclohexylcyclohexylamine (1:1).

LC-MS (Method 1): R _t = 0.87 min; MS (ESIpos): m/z = 737 (M+H) ⁺ .

Intermediate F194

N-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}-L-valyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-L-alaninamide

The title compound was first prepared from Example 98 by coupling with N-[(benzyloxy)carbonyl]-L-valyl-L-alanine in the presence of HATU and N , N -diisopropylethylamine. In the next step, the Z protecting group was removed by hydrogenation over 10% palladium on activated carbon under standard pressure of hydrogen at room temperature for 1 hour, and the deprotected intermediate was then converted to the title compound by reaction with 1,1'-[(1,5-dioxopentan-1,5-diyl)bis(oxy)]dipyrrolidine-2,5-dione as described for Intermediate F58.

LC-MS (Method 1): R _t = 1.19 min; MS (ESIpos): m/z = 851 [M+H] ⁺ .

Intermediate F195

Trifluoroacetic acid/N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyl}-N'-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]succinamide (1:1)

The title compound was prepared by coupling 26 mg (0.035 mmol) of intermediate C65 with 18 mg (0.07 mmol) of commercial trifluoroacetic acid/1-(2-aminoethyl)-1H-pyrrole-2,5-dione (1:1) in 8 ml of DMF in the presence of 40 mg (0.1054 mmol) of HATU and 61 μl of N , N -diisopropylethylamine, and subsequent deprotection with zinc chloride in trifluoroethanol as described for intermediate F119. Purification by preparative HPLC yielded 16 mg (43% of theory over 2 steps) of the title compound.

LC-MS (Method 1): R _t = 0.85 min; MS (ESIpos): m/z = 721 (M+H) ⁺ .

¹ H NMR (500 MHz, DMSO-d ₆ ): d = 7.99 (t, 1H), 7.95 (t, 1H), 7.6-7.75 (m, 4H), 7.5 (s, 1H) 7.2-7.4 (m, 6H), 6.8-7.0 (m, 4H), 5.63 (s, 1H), 4.9 and 5.2 (2d, 2H), 4.26 and 4.0 (2d, 2H), 3.3-3.6 (m, 4H), 3.15-3.25 (m, 3H), 2.85-3.0 (m, 2H), 2.2-2.3 (m, 4H), 0.64 and 1,49 (2m, 2H), 0.81 (s, 9H).

Intermediate F196

Trifluoroacetic acid/N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanine 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl ester (1:1)

First, 15 mg (0.023 mmol) of intermediate C58 was placed in 4 ml of DMF and reacted with 8.2 mg (0.025 mmol) of intermediate L67 in the presence of 13.0 mg (0.034 mmol) of HATU and 16 μl of N , N -diisopropylethylamine. After stirring at room temperature for 30 minutes, the product was concentrated, and the residue was purified by preparative HPLC. After combining the appropriate fractions and evaporating the solvent, the residue was lyophilized from acetonitrile/water (1:1). This yielded 4.3 mg (20% of theory) of the protected intermediate.

LC-MS (Method 1): R _t = 1.35 min; MS (EIpos): m/z = 852 [M+H] ⁺ .

4.3 mg (4.5 μmol) of this intermediate were dissolved in 1 ml of trifluoroethanol and deprotected with 3.65 mg (27 μmol) of zinc chloride as described for intermediate F119. Purification by preparative HPLC yielded 1 mg (25% of theory over 2 steps) of the title compound.

LC-MS (Method 1): R _t = 0.88 min; MS (ESIpos): m/z = 708 (M+H) ⁺ .

Intermediate F204

Trifluoroacetic acid/(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-N-(2-{[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propionyl]amino}ethyl)butanamide (1:1)

First, 25 mg (0.038 mmol) of intermediate C58 was reacted with 16.5 mg (75%) (0.038 mmol) of intermediate L68 in the presence of 17 mg (0.046 mmol) of HATU and 20 μl of N , N -diisopropylethylamine. After stirring at room temperature for 60 minutes, the mixture was concentrated, and the residue was purified by preparative HPLC. This yielded 18.3 mg (56% of theory) of the protected intermediate.

LC-MS (Method 1): R _t = 1.32 min; MS (EIpos): m/z = 851 [M+H] ⁺ .

In the second step, this intermediate was dissolved in 3 ml of 2,2,2-trifluoroethanol. 12 mg (0.086 mmol) of zinc chloride was added, and the preparation was stirred at 50°C for 2 hours. Then, 25 mg (0.086 mmol) of ethylenediamine-N,N,N',N'-tetraacetic acid and 2 ml of 0.1% aqueous trifluoroacetic acid were added. The preparation was purified by preparative HPLC. Concentration of the appropriate fractions and lyophilization of the residue from acetonitrile/water yielded 11 mg (62% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.85 min; MS (ESIpos): m/z = 707 (M+H) ⁺ .

Intermediate F205

Trifluoroacetic acid/N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl-L-valyl-N ⁵ -carbamoyl-L-ornithine 1-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]piperidin-4-yl ester (1:1)

The synthesis was carried out by coupling 25 mg (0.034 mmol) of intermediate C61 and 29 mg (0.041 mmol) of intermediate L69 in the presence of HATU and N , N -diisopropylethylamine, followed by hydrogenation with palladium-activated carbon (10%) at standard pressure, then coupling with (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetic acid in the presence of HATU and N , N -diisopropylethylamine, and finally cleavage of the 2-(trimethylsilyl)ethoxycarbonyl protecting group with zinc chloride. HPLC purification yielded 11 mg (26% of theory over 4 steps).

LC-MS (Method 1): R _t = 0.86 min; MS (ESIpos): m/z = 1061 (M+H) ⁺ .

Intermediate F206

Trifluoroacetic acid/N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl-L-valyl-L-alanine 1-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]piperidin-4-yl ester (1:1)

The synthesis was performed analogously to intermediate F205.

LC-MS (Method 1): R _t = 0.86 min; MS (ESIpos): m/z = 975 (M+H) ⁺ .

Intermediate F207

N ⁶ -(N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl)-N ² -{N-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-valyl-L-alanyl}-L-lysine/trifluoroacetic acid (1:1)

The title compound was prepared analogously to intermediate F155.

LC-MS (Method 1): R _t = 0.81 min; MS (ESIpos): m/z = 1020 (M+H) ⁺ .

Intermediate F209

R-{2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}-N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecanoyl]-L-cysteine/trifluoroacetic acid (1:1)

93.9 mg (0.78 mmol) of L-cysteine was suspended in a solution of 93.0 mg (1.11 mmol) of sodium bicarbonate and 0.9 ml of water. 70.0 mg (0.11 mmol) of 2-(trimethylsilyl)ethyl {3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(chloroacetyl)amino]propyl}carbamate (Intermediate C70) was dissolved in 6.0 ml of isopropanol, and 202.3 mg (1.33 mmol) of 1,8-diazabicyclo[5.4.0]undec-7-ene was added. The reaction mixture was stirred at 50°C for 90 minutes. Water (0.1% TFA) was added to the preparation and purified by preparative RP-HPLC (column: Reprosil 125x30; 10µm, flow rate: 50 ml/min, MeCN/water; 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 53.9 mg (59% of theory) of the compound R-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl)-L-cysteine/trifluoroacetic acid (1:1).

LC-MS (Method 1): R _t = 1.24 min; MS (ESIpos): m/z = 717 (M+H) ⁺ .

86.0 mg (0.1 mmol) of R-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl)-L-cysteine/trifluoroacetic acid (1:1) and 58 1.5 mg (0.11 mmol) of 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-{15-[(2,5-dioxopyrrolidin-1-yl)oxy]-15-oxo-3,6,9,12-tetraoxapentadecan-1-yl}propanamide was dissolved in 4.0 mL of DMF, and 20.9 mg (0.21 mmol) of 4-methylmorpholine was added. The reaction mixture was stirred at room temperature overnight. 15.5 mg (0.26 mmol) of HOAc was added, and the reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 125x30; 10µm, flow rate: 50 mL/min, MeCN/water; 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 68.6 mg (59% of theory) of the compound R-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl)-N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecan-1-oyl]-L-cysteine.

LC-MS (Method 6): R _t = 2.88 min; MS (ESIpos): m/z = 1115 (M+H) ⁺ .

46.4 mg (0.04 mmol) of R-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl)-N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecan-1-oyl]-L-cysteine was dissolved in 2.0 ml of trifluoroethanol, and 17.0 mg (0.13 mmol) of zinc dichloride was added. The reaction mixture was stirred at 50°C overnight. An additional 8.5 mg (0.07 mmol) of zinc dichloride was added and the mixture was stirred at 50°C overnight. To the reaction mixture was added 36.5 mg (0.13 mmol) of ethylenediamine-N,N,N',N'-tetraacetic acid, stirred for 10 minutes, and then water (0.1% TFA) was added. Purification was performed directly by preparative RP-HPLC (column: Reprosil 125x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 19.4 mg (43% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.94 min; MS (ESIpos): m/z = 971 (M+H) ⁺ .

Intermediate F210

S-{2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}-N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecanoyl]-D-cysteine/trifluoroacetic acid (1:1)

The title compound was prepared similarly to the synthesis of intermediate F209 using D-cysteine.

LC-MS (Method 1): R _t = 0.91 min; MS (ESIpos): m/z = 971 (M+H) ⁺ .

Intermediate F211

Trifluoroacetic acid/3-({2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}sulfanyl)-N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]propionamide (1:1)

30.0 mg (0.05 mmol) of 2-(trimethylsilyl)ethyl {3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(chloroacetyl)amino]propyl}carbamate (Intermediate C70) was initially charged with 5.5 mg (0.05 mmol) of 3-sulfanylpropionic acid in 0.5 ml of methanol containing one drop of water. 23.0 mg (0.17 mmol) of carbonic acid was then added, and the reaction mixture was stirred at 50°C for 4 hours. Ethyl acetate was added, and the organic phase was washed once with water and once with saturated NaCl solution. The organic phase was dried over magnesium sulfate, and the solvent was evaporated under vacuum. The residue was used in the next step of the synthesis without further purification. This gave 30.3 mg (86% of theory) of the compound 11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-14-thia-7,11-diaza-2-silaheptadecan-17-oic acid.

LC-MS (Method 1): R _t = 1.39 min; MS (ESIpos): m/z = 702 (M+H) ⁺ .

30.0 mg (0.04 mol) of 11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-14-thia-7,11-diaza-2-silaheptadecane-17-oic acid and 9.8 mg (0.06 mmol) of 1-(2-aminoethyl)-1H-pyrrole-2,5-dione hydrochloride (1:1) were initially charged in 2.0 ml of acetonitrile, and 44.2 mg (0.34 mmol) of N , N -diisopropylethylamine were added. 35.4 mg (0.06 mmol) of T3P (50% in ethyl acetate) were added, and the reaction mixture was stirred at room temperature overnight. Water was added and the product was purified directly by preparative RP-HPLC (column: Reprosil 125x30; 10µm, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 22.0 mg (63% of theory) of the compound [2-(trimethylsilyl)ethyl 3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}{[(3-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino}-3-oxopropyl)sulfanyl]acetyl}amino)propyl]carbamate.

LC-MS (Method 1): R _t = 1.41 min; MS (ESIpos): m/z = 824 (M+H) ⁺ .

22.0 mg (0.03 mol) of 2-(trimethylsilyl)ethyl [3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}{[(3-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino}-3-oxopropyl)sulfanyl]acetyl}amino)propyl]carbamate was dissolved in 1.0 ml of trifluoroethanol, and 9.1 mg (0.07 mmol) of zinc dichloride was added. The reaction mixture was stirred at 50°C for 5 hours. 19.5 mg (0.07 mmol) of ethylenediamine-N,N,N',N'-tetraacetic acid was added to the reaction mixture, stirred for 10 minutes, and then water (0.1% TFA) was added. Purify directly by preparative RP-HPLC (column: Reprosil 125x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent is evaporated under vacuum, and the residue is dried under high vacuum. This yields 15.0 mg (71% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.88 min; MS (ESIpos): m/z = 680 (M+H) ⁺ .

Intermediate F212

Trifluoroacetic acid/N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-10-oxo-3,6-dioxa-13-thia-9-azapentadecan-15-amide (1:1)

28.8 mg (0.04 mmol) of 11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-14-thia-7,11-diaza-2-silaheptadecane-17-oic acid (Intermediate C69) were initially loaded in 1.9 ml of acetonitrile together with 18.3 mg (0.05 mmol) of trifluoroacetic acid/1-{2-[2-(2-aminoethoxy)ethoxy]ethyl}-1H-pyrrole-2,5-dione (1:1) (Intermediate L59). Then, 42.4 mg (0.33 mmol) of N , N -diisopropylethylamine and 33.9 mg (0.05 mmol) of T3P (50% in ethyl acetate) were added dropwise. The reaction mixture was stirred at room temperature overnight. The reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 125x30; 10µm, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 10.7 mg (26% of theory) of 2-(trimethylsilyl)ethyl 16-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-10,15-dioxo-3,6-dioxa-13-thia-9,16-diazanonadecan-19-yl]carbamate.

LC-MS (Method 1): R _t = 1.44 min; MS (ESIpos): m/z = 812 (M+H) ⁺ .

10.7 mg (0.01 mol) of 2-(trimethylsilyl)ethyl [16-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-10,15-dioxo-3,6-dioxa-13-thia-9,16-diazanonadecan-19-yl]carbamate was dissolved in 0.8 ml of trifluoroethanol, and 8.0 mg (0.06 mmol) of zinc dichloride was added. The reaction mixture was stirred at 50°C for 5 hours. 17.1 mg (0.06 mmol) of ethylenediamine-N,N,N',N'-tetraacetic acid was added to the reaction mixture, stirred for 10 minutes, and then water (0.1% TFA) was added. Purify directly by preparative RP-HPLC (column: Reprosil 125x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent is evaporated under vacuum and the residue is dried under high vacuum.

LC-MS (Method 1): R _t = 1.03 min; MS (ESIpos): m/z = 768 (M+H) ⁺ .

Intermediate F213

Trifluoroacetic acid/3-({2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}sulfanyl)-N-(2-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}ethyl)propionamide (1:1)

27.5 mg (0.04 mmol) of 11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-14-thia-7,11-diaza-2-silaheptadecane-17-oic acid (Intermediate C69) were initially loaded in 1.8 ml of acetonitrile together with 15.9 mg (0.05 mmol) of trifluoroacetic acid/N-(2-aminoethyl)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamide (1:1) (Intermediate L1). Then, 32.4 mg (0.31 mmol) of N , N -diisopropylethylamine and 32.4 mg (0.05 mmol) of T3P (50% in ethyl acetate) were added dropwise. The reaction mixture was stirred at room temperature overnight. The reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 125x30; 10µm, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 11.9 mg (35% of theory) of 2-(trimethylsilyl)ethyl 13-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,7,12-trioxo-10-thia-3,6,13-triazahexadec-16-yl]carbamate.

LC-MS (Method 1): R _t = 1.39 min; MS (ESIpos): m/z = 881 (M+H) ⁺ .

11.9 mg (0.01 mol) of 2-(trimethylsilyl)ethyl [13-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,7,12-trioxo-10-thia-3,6,13-triazahexadec-16-yl]carbamate was dissolved in 1.0 ml of trifluoroethanol, and 5.5 mg (0.04 mmol) of zinc dichloride was added. The reaction mixture was stirred at 50°C overnight. 11.8 mg (0.04 mmol) of ethylenediamine-N,N,N',N'-tetraacetic acid was added to the reaction mixture, stirred for 10 minutes, and then water (0.1% TFA) was added. Purify directly by preparative RP-HPLC (column: Reprosil 125x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent is evaporated under vacuum, and the residue is dried under high vacuum. This yields 7.4 mg (60% of theory) of the title compound.

LC-MS (Method 5): R _t = 2.75 min; MS (ESIpos): m/z = 737 (M+H) ⁺ .

Intermediate F214

N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecanoyl]-L-α-glutamyl-S-{2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}-L-cysteine/trifluoroacetic acid (1:1)

111.7 mg (0.30 mmol) of (2S)-5-(benzyloxy)-2-{[(benzyloxy)carbonyl]amino}-5-oxopentanoic acid was initially charged in 3.0 ml of DMF, and 46.1 g (0.30 mmol) of HOBt, 96.6 mg (0.30 mmol) of TBTU, and 38.9 mg (0.30 mmol) of N , N -diisopropylethylamine were added. The reaction mixture was stirred at room temperature for 10 minutes. 250.0 mg (0.30 mmol) of S-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl)-L-cysteine/trifluoroacetic acid (1:1) (Intermediate C71) was dissolved in 116.3 mg (0.9 mmol) of N , N -diisopropylethylamine, and 3.0 ml of DMF was added. The reaction mixture was stirred at room temperature overnight. The reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 125x30; 10µm, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 257.0 mg (80% of theory) of the compound (16R)-11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-16-{[(2S)-5-(benzyloxy)-2-{[(benzyloxy)carbonyl]amino}-5-oxopentanoyl]amino}-2,2-dimethyl-6,12-dioxo-5-oxa-14-thia-7,11-diaza-2-silahexadecanoic acid.

LC-MS (Method 1): R _t = 1.55 min; MS (ESIpos): m/z = 1071 (M+H) ⁺ .

Under argon, 24.6 mg (0.11 mmol) of palladium(II) acetate were initially charged in 5.0 ml of dichloromethane and 33.2 mg (0.33 mmol) of triethylamine and 254.3 mg (2.19 mmol) of triethylsilane were added. The reaction mixture was stirred at room temperature for 5 minutes, and 234.1 mg (0.22 mmol) of (16R)-11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-16-{[(2S)-5-(benzyloxy)-2-{[(benzyloxy)carbonyl]amino}-5-oxopentanoyl]amino}-2,2-dimethyl-6,12-dioxo-5-oxa-14-thia-7,11-diaza-2-silaheptadecane-17-oic acid, dissolved in 5.0 ml of dichloromethane, was added. The reaction mixture was stirred at room temperature overnight. The reaction mixture was filtered through a cardboard filter, and the filter cake was washed with dichloromethane. The solvent was evaporated under vacuum without heating. The residue was purified by preparative RP-HPLC (column: Reprosil 250x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 177.5 mg (85% of theory) of the compound L-α-glutamyl-S-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl)-L-cysteine/trifluoroacetic acid (1:1).

LC-MS (Method 1): R _t = 1.07 min; MS (ESIpos): m/z = 846 (M+H) ⁺ .

20.0 mg (20.83 μmol) of L-α-glutamyl-S-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl)-L-cysteine/trifluoroacetic acid (1:1) were initially mixed with 11.8 mg of 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-{15-[(2,5-dioxopyrrolidin-1-yl)oxy]-15-oxo-3,6,9,12-tetraoxapentadecan-1-yl}propanamide (22.91 μmol) was added to 1.5 mL of DMF, along with 6.3 mg (62.49 μmol) of 4-methylmorpholine. The reaction mixture was stirred at room temperature overnight, followed by the addition of 4.4 mg (0.07 mmol) of acetic acid. The reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 250x30; 10 μm, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 19.1 mg (74% of theory) of the compound N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecan-1-oyl]-L-α-glutamyl-S-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl)-L-cysteine.

LC-MS (Method 1): R _t = 1.24 min; MS (ESIpos): m/z = 1244 (M+H) ⁺ .

17.5 mg (14.06 μmol) of N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecan-1-oyl]-L-α-glutamyl-S-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl)-L-cysteine was dissolved in 1.5 ml of trifluoroethanol, and 11.5 mg (84.37 μmol) of zinc dichloride was added. The reaction mixture was stirred at 50°C for 4 hours. To the reaction mixture was added 24.7 mg (0.08 mmol) of ethylenediamine-N,N,N',N'-tetraacetic acid, stirred for 10 minutes, and then water (0.1% TFA) was added. Purification was carried out directly by preparative RP-HPLC (column: Reprosil 125x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 10.8 mg (63% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.89 min; MS (ESIpos): m/z = 1100 (M+H) ⁺ .

Intermediate F215

N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecanoyl]-L-valyl-N-{3-[({[(2R)-2-acetylamino-2-carboxyethyl]sulfanyl}acetyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]propyl}-L-alaninamide

14.9 mg (0.02 mmol) of N-acetyl-S-[2-([3-(L-alanylamino)propyl]{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)-2-oxoethyl]-L-cysteine/trifluoroacetic acid (1:1) (Example 229) and 7.1 mg (0.02 mmol) of N-[(benzyloxy)carbonyl]-L-valine 2,5-dioxopyrrolidin-1-yl ester were initially charged in 1.0 ml of DMF, and 5.7 mg (0.06 mmol) of 4-methylmorpholine were added. The reaction mixture was stirred at room temperature overnight, and then 4.5 mg (0.08 mmol) of acetic acid were added. The reaction mixture was purified directly by preparative RP-HPLC (column: Reprosil 250x30; 10µ, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 13.3 mg (78% of theory) of the compound N-[(benzyloxy)carbonyl]-L-valyl-N-{3-[({[(2R)-2-acetamido-2-carboxyethyl]sulfanyl}acetyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]propyl}-L-alaninamide.

LC-MS (Method 1): R _t = 1.24 min; MS (ESIpos): m/z = 919 (M+H) ⁺ .

11.1 mg (0.01 mmol) of N-[(benzyloxy)carbonyl]-L-valyl-N-{3-[({[(2R)-2-acetamido-2-carboxyethyl]sulfanyl}acetyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]propyl}-L-alaninamide was dissolved in 5.0 ml of ethanol, 1.0 mg of palladium-activated carbon (10%) was added, and hydrogenation was carried out overnight at room temperature and standard pressure. The reaction mixture was filtered through Celite, and the filter cake was washed with an ethanol/THF/water mixture. The solvent was evaporated under vacuum. The residue was purified by preparative RP-HPLC (column: Reprosil 250x30; 10µm, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was lyophilized. This gave 7.5 mg (69% of theory) of the compound L-valyl-N-{3-[({[(2R)-2-acetylamino-2-carboxyethyl]sulfanyl}acetyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]propyl}-L-alaninamide/trifluoroacetic acid (1:1).

LC-MS (Method 1): R _t = 0.86 min; MS (ESIpos): m/z = 785 (M+H) ⁺ .

7.3 mg (8.12 μmol) of L-valyl-N-{3-[({[(2R)-2-acetamido-2-carboxyethyl]sulfanyl}acetyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]propyl}-L-alaninamide/trifluoroacetic acid (1:1) were initially charged with 4.6 mg (8.93 μmol) of 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-{15-[(2,5-dioxopyrrolidin-1-yl)oxy]-15-oxo-3,6,9,12-tetraoxapentadecan-1-yl}propanamide in 0.5 ml of DMF and 2.5 mg (24.36 μmol) of 4-methylmorpholine were added. The reaction mixture was stirred at room temperature overnight, then 4.4 mg (0.03 mmol) of acetic acid was added. The reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 250x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 4.9 mg (50% of theory) of the title compound.

LC-MS (Method 1): R _t = 1.07 min; MS (ESIpos): m/z = 1183 (M+H) ⁺ .

Intermediate F216

S-{2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}-N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecanoyl]-L-cysteinyl-β-alanine/trifluoroacetic acid (1:1)

Under argon, 30.2 mg (0.06 mmol) of N,N'-bis[(benzyloxy)carbonyl]-L-cystine was initially charged in 2.0 mL of water and 2.0 mL of isopropanol, and 56.7 mg (0.20 mmol) of TCEP was added. The reaction mixture was stirred at room temperature for 30 minutes. Then, 50.0 mg (0.08 mmol) of 2-(trimethylsilyl)ethyl {3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(chloroacetyl)amino]propyl}carbamate (Intermediate C70) and 122.2 mg (0.48 mmol) of 1,8-diazabicyclo[5.4.0]undec-7-ene, dissolved in 2.0 mL of isopropanol, were added, and the reaction mixture was stirred at 50°C for 7 hours. An additional 122.2 mg (0.48 mmol) of 1,8-diazabicyclo[5.4.0]undec-7-ene was then added, and the reaction mixture was stirred at 50°C for 1 hour. The mixture was diluted with ethyl acetate, and the organic phase was extracted with water and saturated sodium bicarbonate solution and washed with saturated NaCl solution. The organic phase was dried over magnesium sulfate, and the solvent was evaporated under vacuum. The residue was purified by preparative RP-HPLC (column: Reprosil 250x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 43.1 mg (64% of theory) of the compound S-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl)-N-[(benzyloxy)carbonyl]-L-cysteine.

LC-MS (Method 1): R _t = 1.46 min; MS (ESIpos): m/z = 851 (M+H) ⁺ .

16.5 mg (0.05 mmol) of 4-methylbenzenesulfonic acid/β-alanine benzyl ester (1:1) were initially charged together with 14.0 mg (0.11 mmol) of N , N -diisopropylethylamine in 1.5 ml of acetonitrile. The reaction mixture was stirred at room temperature for 3 minutes, and then 30.8 mg (0.04 mmol) of S-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl)-N-[(benzyloxy)carbonyl]-L-cysteine, 23.4 mg (0.18 mmol) of N , N -diisopropylethylamine, and 29.9 mg (0.05 mmol) of T3P (50% in ethyl acetate) dissolved in 1.5 ml of acetonitrile were added. The reaction mixture was stirred at room temperature overnight. Water was added, and the reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 250x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. The resulting compound was S-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl)-N-[(benzyloxy)carbonyl]-L-cysteinyl-β-alanine benzyl ester.

LC-MS (Method 1): R _t = 1.59 min; MS (ESIpos): m/z = 1012 (M+H) ⁺ .

43.8 mg (43.3 μmol) of S-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl)-N-[(benzyloxy)carbonyl]-L-cysteinyl-β-alanine benzyl ester were dissolved in 8.0 ml of ethanol, 4.4 mg of palladium-activated carbon (10%) were added, and hydrogenation was carried out overnight at room temperature and standard pressure. The reaction mixture was filtered through a cardboard filter, and the filter cake was washed with ethanol. The solvent was evaporated under vacuum. The residue was processed twice more as described above. The residue was purified by preparative RP-HPLC (column: Reprosil 250x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 4.9 mg (50% of theory) of the compound S-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl)-L-cysteinyl-β-alanine/trifluoroacetic acid (1:1).

LC-MS (Method 1): R _t = 1.08 min; MS (ESIpos): m/z = 788 (M+H) ⁺ .

14.5 mg (16.1 μmol) of S-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl)-L-cysteinyl-β-alanine/trifluoroacetic acid (1:1) was added to the mixture. The product was initially charged with 9.1 mg (17.7 μmol) of 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-{15-[(2,5-dioxopyrrolidin-1-yl)oxy]-15-oxo-3,6,9,12-tetraoxapentadecan-1-yl}propanamide in 1.0 mL of DMF and 4.9 mg (48.2 μmol) of 4-methylmorpholine was added. The reaction mixture was stirred at room temperature overnight, after which 3.4 mg (0.06 mmol) of acetic acid was added. The reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 250x30; 10 µm, flow rate: 50 mL/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 4.9 mg (50% of theory) of the compound S-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl)-L-cysteinyl-β-alanine/trifluoroacetic acid (1:1).

LC-MS (Method 1): R _t = 1.28 min; MS (ESIpos): m/z = 1186 (M+H) ⁺ .

14.1 mg (11.9 μmol) of S-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl)-N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecan-1-oyl]-L-cysteinyl-β-alanine/trifluoroacetic acid (1:1) was dissolved in 1.5 ml of trifluoroethanol, and 9.7 mg (71.3 μmol) of zinc dichloride was added. The reaction mixture was stirred at 50°C for 3 hours. An additional 9.7 mg (71.3 μmol) of zinc dichloride was added, and the reaction mixture was stirred at 50°C for 3 hours. Another 9.7 mg (71.3 μmol) of zinc dichloride was added, and the reaction mixture was stirred at 70°C for 4 hours. 20.8 mg (0.07 mmol) of ethylenediamine-N,N,N',N'-tetraacetic acid was added to the reaction mixture, stirred for 10 minutes, and then water (0.1% TFA) was added. Purification was carried out directly by preparative RP-HPLC (column: Reprosil 125x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was lyophilized. This yielded 6.2 mg (44% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.82 min; MS (ESIpos): m/z = 1042 (M+H) ⁺ .

Intermediate F217

S-{2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}-N-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-cysteine/trifluoroacetic acid (1:1)

Under argon, 7.5 mg (0.05 mmol) of (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetic acid was initially charged in 1.5 ml of DMF, and 7.5 mg (0.05 mmol) of HOBt, 15.5 mg (0.05 mmol) of TBTU, and 6.2 mg (0.05 mmol) of N , N -diisopropylethylamine were added. The reaction mixture was stirred at room temperature for 10 minutes. Then, 40.0 mg (0.05 mmol) of S-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl)-L-cysteine dissolved in 1.5 ml of DMF/trifluoroacetic acid (1:1) (Intermediate C71) and 18.7 mg (0.14 mmol) of N , N -diisopropylethylamine were added, and the reaction mixture was stirred at room temperature overnight. The reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 250x30; 10 µm, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 11.2 mg (25% of theory) of the compound S-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl)-N-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-cysteine.

LC-MS (Method 1): R _t = 1.37 min; MS (ESIpos): m/z = 854 (M+H) ⁺ .

10.9 mg (12.8 μmol) of S-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl)-N-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-cysteine was dissolved in 2.0 ml of trifluoroethanol, and 10.4 mg (76.6 μmol) of zinc dichloride was added. The reaction mixture was stirred at 50°C for 4 hours. 22.4 mg (0.08 mmol) of ethylenediamine-N,N,N',N'-tetraacetic acid was added to the reaction mixture, which was stirred for 10 minutes, and then water (0.1% TFA) was added. Purify directly by preparative RP-HPLC (column: Reprosil 250x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent is evaporated under vacuum and the residue is lyophilized. This yields 7.5 mg (65% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.92 min; MS (ESIpos): m/z = 710 (M+H) ⁺ .

Intermediate F218

N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecanoyl]-L-γ-glutamyl-S-{2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}-L-cysteine/trifluoroacetic acid (1:1)

Under argon, 22.9 mg (0.06 mmol) of (4S)-5-(benzyloxy)-4-{[(benzyloxy)carbonyl]amino}-5-oxopentanoic acid was initially charged in 2.0 ml of DMF, and 9.4 mg (0.05 mmol) of HOBt, 19.8 mg (0.06 mmol) of TBTU, and 8.0 mg (0.06 mmol) of N , N -diisopropylethylamine were added. The reaction mixture was stirred at room temperature for 10 minutes. Then, 51.2 mg (0.06 mmol) of S-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl)-L-cysteine (Intermediate C71) and 23.9 mg (0.19 mmol) of N , N -diisopropylethylamine, dissolved in 1.0 ml of DMF, were added, and the reaction mixture was stirred at room temperature overnight. The reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 250x30; 10 µm, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 11.2 mg (25% of theory) of the compound (16R)-11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-16-{[(4S)-5-(benzyloxy)-4-{[(benzyloxy)carbonyl]amino}-5-oxopentanoyl]amino}-2,2-dimethyl-6,12-dioxo-5-oxa-14-thia-7,11-diaza-2-silahexadecanoic acid.

LC-MS (Method 1): R _t = 1.52 min; MS (ESIpos): m/z = 1070 (M+H) ⁺ .

Under argon, 3.9 mg (0.02 mmol) of palladium(II) acetate were initially charged in 1.0 ml of dichloromethane and 5.3 mg (0.05 mmol) of triethylamine and 254.3 mg (2.19 mmol) of triethylsilane were added. The reaction mixture was stirred at room temperature for 5 minutes, and 18.6 mg (0.02 mmol) of (16R)-11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-16-{[(4S)-5-(benzyloxy)-4-{[(benzyloxy)carbonyl]amino}-5-oxopentanoyl]amino}-2,2-dimethyl-6,12-dioxo-5-oxa-14-thia-7,11-diaza-2-silaheptadecanoic acid dissolved in 1.0 ml of dichloromethane was added. The solvent was evaporated under vacuum without heating. The residue was taken up in acetonitrile, filtered through a syringe filter, and purified by preparative RP-HPLC (column: Reprosil 250x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 11.0 mg (66% of theory) of the compound L-γ-glutamyl-S-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl)-L-cysteine/trifluoroacetic acid (1:1).

LC-MS (Method 1): R _t = 1.14 min; MS (ESIpos): m/z = 846 (M+H) ⁺ .

15.0 mg (15.6 μmol) of L-γ-glutamyl-S-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl)-L-cysteine/trifluoroacetic acid (1:1 ) was initially charged with 8.8 mg (17.2 μmol) of 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-{15-[(2,5-dioxopyrrolidin-1-yl)oxy]-15-oxo-3,6,9,12-tetraoxapentadecan-1-yl}propanamide in 1.0 mL of DMF and 4.7 mg (46.9 μmol) of 4-methylmorpholine was added. The reaction mixture was stirred at room temperature overnight, after which 3.3 mg (0.06 mmol) of acetic acid was added. The reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 250x30; 10 µm, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 14.2 mg (70% of theory) of the compound N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecan-1-oyl]-L-γ-glutamyl-S-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl)-L-cysteine.

LC-MS (Method 4) R _t = 1.24 min; MS (ESIpos): m/z = 1244 (M+H) ⁺ .

13.8 mg (11.1 μmol) of N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecan-1-oyl]-L-γ-glutamyl-S-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl)-L-cysteine was dissolved in 2.0 ml of trifluoroethanol, and 9.1 mg (66.5 μmol) of zinc dichloride was added. The reaction mixture was stirred at 50°C for 4 hours. To the reaction mixture was added 19.4 mg (0.07 mmol) of ethylenediamine-N,N,N',N'-tetraacetic acid, stirred for 10 minutes, and then water (0.1% TFA) was added. Purification was carried out directly by preparative RP-HPLC (column: Reprosil 250x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 6.9 mg (50% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.89 min; MS (ESIpos): m/z = 1100 (M+H) ⁺ .

Intermediate F235

Trifluoroacetic acid/N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecanoyl]-L-valyl-N-{4-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}carbamoyl]phenyl}-L-alaninamide (1:1)

120.0 mg (0.22 mmol) of 2-(trimethylsilyl)ethyl 3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)propyl]carbamate (see Synthesis of Intermediate C11) and 52.1 mg (0.28 mmol) of 4-nitrobenzoyl chloride were dissolved in 8.0 ml of dichloromethane, and 28.4 mg (0.28 mmol) of triethylamine were added. The reaction mixture was stirred at room temperature overnight. The solvent was evaporated under vacuum, and the residue was purified by preparative RP-HPLC (column: Reprosil 125x30; 10µm, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). This gave 97.7 mg (64% of theory) of the compound 2-(trimethylsilyl)ethyl 3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(4-nitrobenzoyl)amino]propyl}carbamate.

LC-MS (Method 1): R _t = 1.54 min; MS (ESIpos): m/z = 705 (M+H) ⁺ .

97.0 mg (0.14 mmol) of 2-(trimethylsilyl)ethyl {3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(4-nitrobenzoyl)amino]propyl}carbamate was dissolved in 5.0 ml of ethanol, 9.7 mg of palladium-activated carbon (10%) was added, and hydrogenation was carried out under standard pressure for 5 hours. The reaction mixture was filtered through a cardboard filter, and the filter cake was washed with ethanol. The solvent was evaporated under vacuum. The residue was used in the next step of the synthesis without further purification. This gave 87.4 mg (88% of theory) of the compound 2-(trimethylsilyl)ethyl {3-[(4-aminobenzoyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]propyl}carbamate.

LC-MS (Method 1): R _t = 1.47 min; MS (ESIpos): m/z = 675 (M+H) ⁺ .

59.3 mg (0.09 mmol) of 2-(trimethylsilyl)ethyl 3-[(4-aminobenzoyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]propyl}carbamate and 25.5 mg (0.11 mmol) of N-[(benzyloxy)carbonyl]-L-alanine were initially charged in 5.0 ml of acetonitrile along with 68.1 mg (0.53 mmol) of N , N -diisopropylethylamine. 72.7 mg (0.11 mmol) of T3P (50% in ethyl acetate) were slowly added. The reaction mixture was stirred at room temperature overnight. The reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 125x30; 10µm, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 52.2 mg (68% of theory) of the compound [(2S)-1-{[4-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}[3-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)propyl]carbamoyl)phenyl]amino}-1-oxopropan-2-yl]carbamic acid benzyl ester].

LC-MS (Method 1): R _t = 1.48 min; MS (ESIpos): m/z = 880 (M+H) ⁺ .

23.9 mg (0.03 mmol) of benzyl [(2S)-1-{[4-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}[3-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)propyl]carbamoyl)phenyl]amino}-1-oxopropan-2-yl]carbamate was dissolved in 3.0 ml of ethyl acetate, 2.4 mg of palladium-activated carbon (10%) was added, and hydrogenation was carried out under standard pressure for 2 hours. The reaction mixture was filtered through a paper filter, and the filter cake was washed with ethyl acetate. The solvent was evaporated under vacuum. The residue was used in the next step of the synthesis without further purification. This gave 20.1 mg (90% of theory) of the compound 2-(trimethylsilyl)ethyl [3-([4-(L-alanylamino)benzoyl]{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)propyl]carbamate.

LC-MS (Method 1): R _t = 1.13 min; MS (ESIpos): m/z = 746 (M+H) ⁺ .

20.0 mg (0.03 mmol) of 2-(trimethylsilyl)ethyl [3-([4-(L-alanylamino)benzoyl]{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)propyl]carbamate were initially charged with 14.9 mg (0.04 mmol) of N-[(benzyloxy)carbonyl]-L-valine 2,5-dioxopyrrolidin-1-yl ester in 2.0 ml of DMF, and 5.4 mg (0.05 mmol) of 4-methylmorpholine were added. The reaction mixture was purified directly by preparative RP-HPLC (column: Reprosil 125x30; 10µm, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gives the compound N-[(benzyloxy)carbonyl]-L-valyl-N-[4-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}[3-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)propyl]carbamoyl)phenyl]-L-alaninamide.

LC-MS (Method 1): R _t = 1.49 min; MS (ESIpos): m/z = 979 (M+H) ⁺ .

17.0 mg (17.4 μmol) of N-[(benzyloxy)carbonyl]-L-valyl-N-[4-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}[3-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)propyl]carbamoyl)phenyl]-L-alaninamide were dissolved in 2.5 ml of ethyl acetate, 1.7 mg of palladium-activated carbon (10%) were added, and hydrogenation was carried out overnight under standard pressure. The reaction mixture was filtered through a paper filter, and the filter cake was washed with ethyl acetate. The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 15.3 mg (60% of theory) of the compound L-valyl-N-[4-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}[3-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)propyl]carbamoyl)phenyl]-L-alaninamide.

LC-MS (Method 1): R _t = 1.15 min; MS (ESIpos): m/z = 845 (M+H) ⁺ .

15.3 mg (0.01 mmol) of L-valyl-N-[4-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}[3-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)propyl]carbamoyl)phenyl]-L-alaninamide were initially charged with 7.9 mg (0.02 mmol) of 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-{15-[(2,5-dioxopyrrolidin-1-yl)oxy]-15-oxo-3,6,9,12-tetraoxapentadecan-1-yl}propanamide in 2.4 ml of DMF and 1.9 mg (0.02 mmol) of 4-methylmorpholine were added. The reaction mixture was stirred at room temperature overnight, then 1.4 mg (0.02 mmol) of acetic acid was added. The reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 125x30; 10µ, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 11.7 mg (70% of theory) of the compound N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecan-1-oyl]-L-valyl-N-[4-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}[3-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)propyl]carbamoyl)phenyl]-L-alaninamide.

11.7 mg (0.01 mmol) of N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecan-1-oyl]-L-γ-glutamyl-S-(11-{(1R)-1-[1-N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecan-1-oyl]-L-valyl-N-[4-({(1R)- 1-[1-Benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}[3-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)propyl]carbamoyl)phenyl]-L-alaninamide was dissolved in 2.0 ml of trifluoroethanol and 3.9 mg (0.03 mmol) of zinc dichloride was added. The reaction mixture was stirred at 50°C overnight. 8.3 mg (0.03 mmol) of ethylenediamine-N,N,N',N'-tetraacetic acid was added to the reaction mixture and stirred for 10 minutes, followed by the addition of water (0.1% TFA). Purify directly by preparative RP-HPLC (column: Reprosil 125x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). Evaporate the solvent under vacuum, and dry the residue under high vacuum. This yields 5.4 mg (47% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.94 min; MS (ESIpos): m/z = 1100 (M+H) ⁺ .

Intermediate F236

(2R)-2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)-4-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}butyric acid/trifluoroacetic acid (1:1)

The synthesis of the title compound was carried out analogously to intermediate F192 from (2R)-4-{[(benzyloxy)carbonyl]amino}-2-[(tert-butoxycarbonyl)amino]butanoic acid/N-cyclohexylcyclohexylamine (1:1).

LC-MS (Method 4): R _t = 1.1 min; MS (ESIpos): m/z = 751 (M+H) ⁺ .

Intermediate F238

Trifluoroacetic acid/N-{(2S)-1-amino-3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propan-2-yl}-N'-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]succinamide (1:1)

18 mg (0.025 mmol) of intermediate C72 was placed in 6 ml of DMF and coupled with 7.5 mg (0.03 mmol) of trifluoroacetic acid/1-(2-aminoethyl)-1H-pyrrole-2,5-dione (1:1) in the presence of 11.3 mg (0.03 mmol) of HATU and 22 μl of N , N -diisopropylethylamine. After stirring at room temperature for 1 hour, the preparation was concentrated and the residue was purified by preparative HPLC. The appropriate fractions were concentrated and the residue was lyophilized from acetonitrile/water (1:1). This yielded 15 mg (67% of theory) of the intermediate.

LC-MS (Method 4): R _t = 1.71 min; MS (EIpos): m/z = 873 [M+Na] ⁺ .

The title compound was then prepared from this intermediate by deprotection with zinc chloride in 4 ml of trifluoroethanol as described for intermediate F119. Purification by preparative HPLC yielded 8.5 mg (63% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.86 min; MS (ESIpos): m/z = 707 (M+Na) ⁺ .

Intermediate F241

Trifluoroacetic acid/(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-N-(2-{[N-(bromoacetyl)glycyl]amino}ethyl)butanamide (1:1)

The title compound was prepared analogously from intermediate C66 by coupling with commercially available 1-(2-bromoacetoxy)pyrrolidine-2,5-dione and subsequent deblocking with zinc chloride.

LC-MS (method 1): R _t = 0.84 min; MS (EIpos): m/z = 733 and 735 [M+H] ⁺ .

Intermediate F242

Trifluoroacetic acid/(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-N-(3-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}propyl)butanamide (1:1)

The synthesis of the title compound was carried out analogously to intermediate F104.

LC-MS (Method 1): R _t = 0.84 min; MS (ESIpos): m/z = 707 (M+H) ⁺ .

Intermediate F243

Trifluoroacetic acid/(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-N-[2-(2-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}ethoxy)ethyl]butanamide (1:1)

The synthesis of the title compound was carried out analogously to intermediate F242.

LC-MS (Method 1): R _t = 0.81 min; MS (ESIpos): m/z = 737 (M+H) ⁺ .

Intermediate F244

N-{2-[(S-{2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}-L-cysteinyl)amino]ethyl}-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide

100 mg (approximately 0.101 mmol) of S-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl)-N-[3-(trimethylsilyl)propionyl]-L-cysteine (Intermediate C 73) were initially charged in 88 ml of dimethylformamide, and 107 mg (approximately 0.15 mmol) of N-(2-aminoethyl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide (Intermediate L73), 46 mg (0.12 mmol) of HATU and 88 μl (0.50 mmol) were added. The reaction mixture was stirred at room temperature for 15 minutes. Water/dichloromethane was added to the mixture, and the organic phase was then washed with water and brine, dried over magnesium sulfate, concentrated on a rotary evaporator, and dried under high vacuum. The residue was used further without further purification. This yielded 92 mg (59%, 72% purity) of the title compound.

LC-MS (Method 1): R _t = 1.59 min; MS (ESIpos): m/z = 1096 (M+H) ⁺ .

Under argon, 40 mg (0.30 mmol) of zinc chloride was added to a solution of 91 mg (approximately 0.06 mmol) of 2-(trimethylsilyl)ethyl [(9R)-4-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-20-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,10,15-trioxo-9-{[3-(trimethylsilyl)propionyl]amino}-7-thia-4,11,14-triazaeicosan-1-yl]carbamate in 1.45 ml of trifluoroethanol. The reaction mixture was stirred at 50° C. for 2 hours. 30 mg (0.22 mmol) of zinc chloride was then added, and the mixture was stirred at room temperature for an additional hour. After adding 52 mg (0.18 mmol) of EDTA and stirring at room temperature for 10 minutes, the mixture was diluted slightly with water/acetonitrile and purified by preparative HPLC (eluent: ACN/water + 0.1% TFA, gradient). This gave 17 mg (31%) of the title compound.

LC-MS (Method 1): R _t = 0.80 min; MS (ESIpos): m/z = 808 (M+H) ⁺ .

Intermediate F245

Trifluoroacetic acid/N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyl}-N'-(2-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}ethyl)succinamide (1:1)

The title compound was prepared by coupling 10 mg (0.0135 mmol) of intermediate C65 with 8 mg (0.027 mmol) of intermediate L1 in 8 ml of DMF in the presence of 15 mg (0.04 mmol) of HATU and 9 μl of N , N -diisopropylethylamine, and subsequent deprotection with zinc chloride in trifluoroethanol as described for intermediate F119. Purification by preparative HPLC yielded 8.8 mg (58% of theory over 2 steps) of the title compound.

LC-MS (Method 1): R _t = 0.84 min; MS (ESIpos): m/z = 778 (M+H) ⁺ .

Intermediate F247

Trifluoroacetic acid/methyl 4-[(2-{[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]amino}-2-oxoethyl)amino]-2-bromo-4-oxobutanoate (1:1)

14 mg (0.018 mmol) of intermediate C66 was dissolved in 14 ml of DCM, and 10.1 mg (0.037 mmol) of 2-bromo-1-ethylpyridinium tetrafluoroborate (BEP) and a total of 250 μl of pyridine were added portionwise, with the pH being maintained between 5 and 6. The pH was then adjusted to 4 with acetic acid. The preparation was concentrated, and the residue was purified by preparative HPLC. Combining the appropriate fractions, lyophilizing, and drying yielded 4 mg (21% of theory) of the protected intermediate, which was subsequently deprotected at the amino function using zinc chloride. HPLC purification and lyophilization yielded 3 mg (72% of theory) of the title compound as a colorless foam.

LC-MS (method 1): R _t = 0.88 min; MS (ESIpos): m/z = 805 and 807 (M+H) ⁺ .

Intermediate F248

Trifluoroacetic acid/(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-N-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy]ethyl}butanamide (1:1)

The title compound was prepared by coupling 10 mg (0.015 mmol) of intermediate C58 with 5 mg (0.017 mmol) of intermediate L12 in the presence of HATU and subsequent deprotection with zinc chloride. This gave 6.5 mg (52% of theory over 2 steps) of the title compound.

LC-MS (Method 1): R _t = 0.91 min; MS (ESIpos): m/z = 680 (M+H) ⁺ .

Intermediate F254

Trifluoroacetic acid/methyl (3S)-4-[(2-{[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butanoyl}amino)ethyl]amino}-2-oxoethyl)amino]-3-bromo-4-oxobutanoate (1:1)

The title compound was prepared analogously to intermediate 247 by coupling 15 mg (0.02 mmol) of intermediate C66 with 21 mg (0.099 mmol) of (2S)-2-bromo-4-methoxy-4-oxobutanoic acid synthesized from (2S)-2-amino-4-methoxy-4-oxobutanoic acid hydrochloride (1:1) as described in (J. Org. Chem. 200, 65, 517-522).

LC-MS (method 1): R _t = 0.89 min; MS (ESIpos): m/z = 805 and 807 (M+H) ⁺ .

1661398 Instruction Manual Part 2

B: Preparation of Antibody/Active Substance Conjugates (ADCs)

B-1. General method for generating anti-TWEAKR antibodies

For example, anti-TWEAKR antibodies were generated by screening phage display libraries of recombinant human TWEAKR SEQ ID NO: 138 and murine TWEAKR SEQ ID NO: 137. The antibodies thus obtained were reformatted into human IgG1 format and used in the examples described herein. In addition, antibodies that bind to TWEAKR are known to those skilled in the art, see, for example, WO2009/020933 (A2) or WO2009140177 (A2).

SEQ ID NO:138 (polypeptide):

SEQ ID NO:137 (polypeptide):

B-2. General Methods for Expressing Anti-TWEAKR Antibodies in Mammalian Cells

Antibodies, such as TPP-2090, are produced in short-term mammalian cell culture as described in Chapter 12 in Tom et al., Methods Express: Expression Systems, ed. Michael R. Dyson and Yves Durocher, Scion Publishing Ltd, 2007 (see AK - Example 1).

B-3. General Methods for Purifying Antibodies from Cell Supernatants

Antibodies, such as TPP-2090, are obtained from cell culture supernatants. The cell supernatants are clarified by cell centrifugation. The cell supernatants are then purified by affinity chromatography on a MabSelect Sure (GE Healthcare) column. To this end, the column is equilibrated in DPBS pH 7.4 (Sigma/Aldrich), the cell supernatant is applied, and the column is washed with approximately 10 column volumes of DPBS pH 7.4 + 500 mM sodium chloride. The antibodies are eluted in 50 mM sodium acetate pH 3.5 + 500 mM sodium chloride and then further purified by gel permeation chromatography on a Superdex 200 column (GE Healthcare) in DPBS pH 7.4.

The commercially available antibody cetuximab (trade name Erbitux) was purified from the commercial product by standard chromatography (Protein A, preparative SEC).

The commercially available antibody trastuzumab (trade name Herceptin) was purified from the commercial product by standard chromatography (Protein A, preparative SEC).

The antibody nimotuzumab was purified from a commercial product (trade name CIMAher) by standard chromatography (Protein A, preparative SEC).

The antibody panitumumab was purified from a commercial product (trade name Vectibix) by standard chromatography (Protein A, preparative SEC).

B-4. General method for coupling to cysteine side chains

The following antibodies were used in this conjugation reaction:

Cetuximab (anti-EGFR AK)

Anti-TWEAKR AK 1 (TPP-2090)

Trastuzumab (anti-Her2 AK)

Nimotuzumab (anti-EGFR AK)

Panitumumab (anti-EGFR AK).

2 to 5 equivalents of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) dissolved in PBS buffer are added to a solution of the corresponding antibody in PBS buffer at a concentration range of 1 mg/ml to 20 mg/ml, preferably approximately 10 mg/ml to 15 mg/ml, and stirred at room temperature for 1 hour. For this purpose, the respective antibody solution can be used at the concentrations indicated in the Examples or, optionally, diluted with PBS buffer to approximately half of the indicated starting concentration to reach the preferred concentration range. Subsequently, depending on the desired loading, 2 to 12 equivalents, preferably approximately 5-10 equivalents, of the maleimide precursor or halide precursor compound to be coupled are added as a solution in DMSO. The amount of DMSO should not exceed 10% of the total volume. The formulation is stirred at room temperature for 60-240 minutes for maleimide precursors and for 8 to 24 hours for halide precursors, then applied to a PD 10 column ( ^Sephadex® G-25, GE Healthcare) equilibrated in PBS and eluted with PBS buffer. Typically, unless otherwise specified, 5 mg of the corresponding antibody in PBS buffer was used for reduction and subsequent coupling. Purification on a PD10 column thus provided a solution of the corresponding ADC in 3.5 ml of PBS buffer in each case. The sample was then concentrated by ultracentrifugation and optionally rediluted with PBS buffer. If necessary, to better separate low molecular weight components, the ultrafiltration concentration was repeated after redilution with PBS buffer. For bioassays, the concentration of the final ADC sample was optionally adjusted to a range of 0.5-15 mg/ml by redilution, if necessary. The protein concentration of each ADC solution shown in the Examples was determined. In addition, the antibody loading (drug/mAb ratio) was determined using the method described under B-7.

Unless otherwise indicated, the immunoconjugates shown in the examples were prepared by this method. Depending on the linker, the ADCs shown in the examples may also optionally be present in the form of hydrolyzed open-chain succinamides attached to the antibody to a lesser or greater extent.

In particular, via the linker substructure

KSP-1-ADC linked to the thiol group of the antibody can also be prepared in a directed manner, optionally via an open-chain succinamide-linked ADC according to Scheme 26, by rebuffering after coupling and stirring at pH 8 for approximately 20 hours.

#1 represents the sulfide bridge to the antibody, and #2 is the connection point to the modified KSP inhibitor.

Such ADCs, in which the linker is hydrolyzed to open the chain succinamide and attached to the antibody, can also be optionally prepared in a directed manner by the following exemplary procedure:

Under argon, a solution of 0.344 mg of TCEP in 100 μL of PBS was added to 60 mg of the antibody in question (c ~12 mg/mL) in 5 mL of PBS. The preparation was stirred at room temperature for 30 minutes, followed by the addition of 0.003 mmol of the maleimide precursor compound dissolved in 600 μL of DMSO. After stirring at room temperature for an additional 1.5 to 2 hours, the preparation was diluted with 1075 μL of PBS buffer pre-adjusted to pH 8.

This solution is then applied to a PD 10 column ( ^Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer, pH 8, and eluted with PBS buffer, pH 8. The eluate is diluted with PBS buffer, pH 8, to a total volume of 14 ml. This solution is stirred overnight at room temperature under argon. Optionally, it is then rebuffered to pH 7.2. The ADC solution is concentrated by ultracentrifugation, rediluted with PBS buffer (pH 7.2), and then optionally reconcentrated to a concentration of approximately 10 mg/ml.

Other potentially hydrolysis-sensitive Thianyl succinimide bridges attached to antibodies in the Examples contain the following linker substructures, where #1 represents the thioether bond to the antibody and #2 represents the point of attachment to the modified KSP inhibitor:

These linker substructures represent the attachment unit to the antibody and (besides the linker composition) have a significant impact on the structure and properties of the metabolites formed in tumor cells.

In the structural formula shown, AK _1A , AK _1B , AK _1E , AK _1I , AK _1H , and AK _1K have the following meanings:

AK _1A = Cetuximab (partially reduced) - S§ ¹

AK _1B = Anti-TWEAKR AK-1 (partially reduced) - S§ ¹

AK _1E = Trastuzumab (partially reduced) - S§ ¹

AK _1I = Nimotuzumab (partially reduced) - S§ ¹

AK _1H = Panitumumab (partially reduced) - S§ ¹

in

§ ¹ represents a bond to a succinimide group or to an isomeric hydrolyzed open-chain succinamide or alkylene group derived therefrom,

and

S represents the sulfur atom of the cysteine residue of the partially reduced antibody.

B-5. General method for coupling to lysine side chain

The following antibodies were used in this conjugation reaction:

Cetuximab (anti-EGFR AK)

Anti-TWEAKR AK 1 (TPP-2090)

Trastuzumab (anti-Her2 AK)

Nimotuzumab (anti-EGFR AK)

Panitumumab (anti-EGFR AK).

Depending on the desired loading, 2 to 8 equivalents of the precursor compound to be coupled are added as a solution in DMSO to a solution of the corresponding antibody in PBS buffer at a concentration ranging from 1 mg/ml to 20 mg/ml, preferably approximately 10 mg/ml. After stirring at room temperature for 30 minutes to 6 hours, the same amount of the precursor compound in DMSO is added. The amount of DMSO should not exceed 10% of the total volume. After stirring at room temperature for an additional 30 minutes to 6 hours, the formulation is applied to a PD 10 column ( ^Sephadex® G-25, GE Healthcare) equilibrated with PBS and eluted with PBS buffer. Typically, unless otherwise specified, 5 mg of the corresponding antibody in PBS buffer is used for reduction and subsequent coupling. Purification on the PD 10 column thus provides a solution of the corresponding ADC in 3.5 ml of PBS buffer in each case. This is then concentrated by ultracentrifugation, and the sample is optionally diluted with PBS buffer. If necessary, to better separate low-molecular-weight components, the concentration by ultrafiltration is repeated after dilution with PBS buffer. For bioassays, the concentration of the final ADC sample was optionally adjusted to the range of 0.5-15 mg/ml by redilution, if necessary.

The protein concentrations of the ADC solutions shown in the examples were determined. In addition, the antibody loading (drug/mAb ratio) was determined using the method described under B-7.

In the structural formula shown, AK _2A , AK _2B , AK _2G , AK _2E , AK _2I , AK _2H , and AK _2K have the following meanings:

AK _2A = Cetuximab (anti-EGFR AK)-NH§ ²

AK _2B = Anti-TWEAKR AK-1- NH§ ²

AK _2E = Trastuzumab-NH§ ²

AK _2I = Nimotuzumab-NH§ ²

AK _2H = panitumumab-NH§ ²

in

§ ² represents the bond to the carbonyl group

and

NH represents the side chain amino group of a lysine residue of the antibody.

B-6a. General method for preparing blocked succinimide-cysteine adducts:

In an exemplary embodiment, 10 micromoles of the maleimide precursor compound described above is placed in 3-5 ml of DMF and 2.1 mg (20 micromoles) of L-cysteine is added. The reaction mixture is stirred at room temperature for 2 to 24 hours, then concentrated under vacuum and purified by preparative HPLC.

B-6aa. General method for preparing isomeric open succinamide-cysteine adducts:

In an exemplary embodiment, 68 micromoles of the maleimide precursor compound described above are placed in 15 ml of DMF, and 36 mg (136 micromoles) of N-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-cysteine is added. The reaction mixture is stirred at room temperature for ~20 hours, then concentrated under vacuum and purified by preparative HPLC. After combining the appropriate fractions and evaporating the solvent under vacuum, the residue is dissolved in 15 ml of THF/water (1:1). 131 microliters of 2M aqueous lithium hydroxide solution are added, and the formulation is stirred at room temperature for 1 hour. The formulation is then neutralized with 1M hydrochloric acid, the solvent evaporated under vacuum, and the residue purified by preparative HPLC. This yields the regioisomeric protected intermediate as a colorless foam at ~50% of theory.

In the final step, 0.023 mmol of these regioisomeric hydrolysis products was dissolved in 3 ml of 2,2,2-trifluoroethanol. 12.5 mg (0.092 mmol) of zinc chloride was added, and the preparation was stirred at 50°C for 4 hours. Then, 27 mg (0.092 mmol) of ethylenediamine-N,N,N',N'-tetraacetic acid was added, and the solvent was evaporated under vacuum. The residue was purified by preparative HPLC. Concentration of the appropriate fractions and lyophilization of the residue from acetonitrile/water yielded the hydrolyzed open sulfanylsuccinamide as a mixture of regioisomers.

B-6b. General method for preparing lysine adducts:

In an exemplary embodiment, 10 micromoles of the activated ester precursor compound described above are placed in 3-5 ml of DMF, and α-amino-protected L-lysine is added in the presence of 30 micromoles of N , N -diisopropylethylamine. The reaction mixture is stirred at room temperature for 2 to 24 hours, then dried under vacuum and purified by preparative HPLC. The protecting groups are then removed by known methods.

Further purification and characterization of the conjugate of the present invention

After the reaction, in some cases, the reaction mixture is concentrated, for example, by ultrafiltration, and then desalted and purified by chromatography, for example, using a Sephadex® G-25 column. Elution is performed, for example, with phosphate-buffered saline (PBS). The solution is then sterile filtered and frozen. Alternatively, the conjugate can be lyophilized.

B-7. Determination of Antibody, Toxin Cluster Loading, and Open Cysteine Adduct Content

After deglycosylation and/or denaturation, in addition to molecular weight determination, for identification of proteins, trypsin digestion was performed, which confirmed the identity of the proteins via tryptic peptides found after denaturation, reduction and derivatization.

The resulting solution of the conjugate described in the examples in PBS buffer was assayed for cluster loading as follows:

The cluster loading of lysine-linked ADCs was determined by mass spectrometric determination of the molecular weight of each conjugate species. The antibody conjugates were first deglycosylated using PNGaseF. The samples were acidified and separated/desalted by HPLC before analysis by mass spectrometry using ESI-MicroTofQ (Bruker Daltonik). All spectra from the signals in the TIC (total ion chromatogram) were summed and the molecular weights of the different conjugate species were calculated using MaxEnt deconvolution. The DAR (drug/antibody ratio) was then calculated after integrating the signals for the different species.

The toxicity of cysteine-linked conjugates was determined by reverse-phase chromatography of reduced and denatured ADCs. Guanidine hydrochloride (GuHCl) (28.6 mg) and DL-dithiothreitol (DTT) solution (500 mM, 3 μL) were added to an ADC solution (1 mg/mL, 50 μL). The mixture was incubated at 55°C for 1 hour and analyzed by HPLC.

HPLC analysis was performed on an Agilent 1260 HPLC system with detection at 220 nm. A Polymer Laboratories PLRP-S polymeric reversed-phase column (Cat. No. PL1912-3802) (2.1 x 150 mm, 8-μm particle size, 1000 Å) was used at a flow rate of 1 ml/min with the following gradient: 0 min, 25% B; 3 min, 25% B; 28 min, 50% B. Eluent A consisted of 0.05% trifluoroacetic acid (TFA) in water, and eluent B consisted of 0.05% TFA in acetonitrile.

The detected peaks were assigned values by comparison with the retention times of the light chain (L0) and heavy chain (H0) of the unconjugated antibody. Peaks detected only in the conjugated sample were designated as light chain with one toxic cluster (L1) and heavy chain with one, two, and three toxic clusters (H1, H2, H3).

The average load of antibody containing clusters was calculated from the peak areas determined by integration as twice the sum of the cluster number-weighted integration results for all peaks divided by the sum of the single-weighted integration results for all peaks. In individual cases, the cluster load may not be accurately determined due to coelution of some peaks.

In cases where the light and heavy chains could not be adequately separated by HPLC, determination of the toxic load of the cysteine-linked conjugates was performed by mass spectrometric determination of the molecular weight of each conjugate species at the light and heavy chains.

Guanidine hydrochloride (GuHCl) (28.6 mg) and DL-dithiothreitol (DTT) solution (500 mM, 3 μL) were added to an ADC solution (1 mg/ml, 50 μL). The mixture was incubated at 55°C for 1 hour and analyzed by mass spectrometry after online desalting using an ESI-MicroTof _Q (Bruker Daltonik).

For DAR determination, all spectra across the signal in the TIC (total ion chromatogram) were summed and the molecular weights of the different conjugate species at the light and heavy chains were calculated based on MaxEnt deconvolution. The average load of antibody containing clusters was calculated by integrating the areas of certain molecular weights as twice the sum of the cluster-weighted integration results of all peaks divided by the sum of the single-weighted integration results of all peaks.

To determine the content of open cysteine adducts, the molecular weight area ratio of closed and open cysteine adducts (MW Δ18 Da) was determined for all singly coupled light and heavy chain variants. The average value of all variants gave the content of open cysteine adducts.

B-8. Examination of Antigen Binding of ADC

After conjugation, the ability of the conjugate to bind to the target molecule is examined. Those skilled in the art are familiar with various methods available for this purpose; for example, the affinity of the conjugate can be examined using ELISA techniques or surface plasmon resonance analysis (BIAcore™ measurement). Those skilled in the art can measure the concentration of the conjugate using conventional methods, such as protein assays for antibody conjugates (see also Doronina et al.; Nature Biotechnol. 2003; 21: 778-784 and Polson et al., Blood 2007; 1102: 616-623).

Example ADC

Example 1A

Here, 70 mg of cetuximab (c=5.1 mg/ml) in PBS were used for coupling to intermediate F1, and the formulation was concentrated by ultracentrifugation after Sephadex purification, rediluted with PBS and reconcentrated.

Protein concentration: 10.8 mg/ml

Drug/mAb ratio: 2.6.

parenthesis

In Example 1A, a compound of the formula wherein n=2.6 was obtained

and a certain antibody (cetuximab). However, the present invention provides not only this specific conjugate having exactly this antibody at this active substance/antibody ratio, but also other binding conjugates of this formula, i.e., conjugates in which cetuximab is replaced by a different antibody or a derivative thereof (such as cysteine) and n=2.6 is replaced by any value within the range of n=1 to 20, preferably n=1-10.

This applies correspondingly to the following examples.

Example 2A

Here, 80 mg of cetuximab (c = 5.1 mg/ml) in PBS was used for coupling to intermediate F2, and the formulation was concentrated by ultracentrifugation after purification on a dextran gel, rediluted with PBS, and reconcentrated. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 11.0 mg/ml

Drug/mAb ratio: 2.5.

Example 2B

Here, 50 mg of anti-TWEAKR AK-1 (c = 10.1 mg/ml) in PBS was used for coupling with intermediate F2, and the preparation was concentrated by ultracentrifugation after purification on a dextran gel, rediluted with PBS, and reconcentrated. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 11.7 mg/ml

Drug/mAb ratio: 3.7.

Example 2E

Here, 5 mg of trastuzumab (c = 13.4 mg/ml) in PBS was used for coupling to intermediate F2, and the formulation was concentrated by ultracentrifugation after purification on a dextran gel and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.77 mg/ml

Drug/mAb ratio: 2.6.

Example 3A

Here, 5 mg of cetuximab (c = 5.1 mg/ml) in PBS was used for coupling to intermediate F3, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 2.87 mg/ml

Drug/mAb ratio: 2.0.

Example 3B

Here, 5 mg of anti-TWEAKR AK-1 (c=16.5 mg/ml) in PBS were used for coupling to intermediate F3, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 2.13 mg/ml

Drug/mAb ratio: 2.1.

Example 4A

Here, 5 mg of cetuximab (c = 5.1 mg/ml) in PBS was used for coupling to intermediate F4, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 2.11 mg/ml

Drug/mAb ratio: 2.4.

Example 4B

Here, 5 mg of anti-TWEAKR AK-1 (c = 16.5 mg/ml) in PBS were used for coupling to intermediate F4, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.69 mg/ml

Drug/mAb ratio: 2.9.

Example 5A

Here, 5 mg of cetuximab (c = 5.1 mg/ml) in PBS was used for coupling to intermediate F5, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.16 mg/ml

Drug/mAb ratio: 2.1.

Example 5B

Here, 5 mg of anti-TWEAKR AK-1 (c = 16.5 mg/ml) in PBS was used for coupling to intermediate F5, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.5 mg/ml

Drug/mAb ratio: 2.6.

Example 6A

Here, 5 mg of cetuximab (c = 5.1 mg/ml) in PBS was used for coupling to intermediate F6, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.24 mg/ml

Drug/mAb ratio: 2.3.

Example 6B

Here, 5 mg of anti-TWEAKR AK-1 (c = 16.5 mg/ml) in PBS was used for coupling to intermediate F6, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.88 mg/ml

Drug/mAb ratio: 2.1.

Example 7A

Here, 5 mg of cetuximab (c = 5.9 mg/ml) in PBS was used for coupling to intermediate F7, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.46 mg/ml

Drug/mAb ratio: 2.9.

Example 7B

Here, 40 mg of anti-TWEAKR AK-1 (c = 12.9 mg/ml) in PBS was used for coupling with intermediate F7, and the preparation was concentrated by ultracentrifugation after dextran gel purification, rediluted with PBS, and reconcentrated. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 11.27 mg/ml

Drug/mAb ratio: 3.0.

Example 8A

Here, 5 mg of cetuximab (c = 12.7 mg/ml) in PBS was used for coupling to intermediate F8, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.91 mg/ml

Drug/mAb ratio: 3.6.

Example 8B

Here, 40 mg of anti-TWEAKR AK-1 (c = 12.9 mg/ml) in PBS were used for coupling to intermediate F8, and the preparation was concentrated by ultracentrifugation after Sephadex purification, rediluted with PBS and reconcentrated.

Protein concentration: 11.54 mg/ml

Drug/mAb ratio: 2.9.

Example 8E

Here, 5 mg of trastuzumab (c = 13.4 mg/ml) in PBS was used for coupling to intermediate F8, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.97 mg/ml

Drug/mAb ratio: 3.5.

Example 8H

Here, 5.0 mg of panitumumab (c = 10 mg/ml) in PBS was used for coupling with intermediate F8. The reduction time with TCEP was increased to 4 hours, and the stirring time for ADC coupling was increased to 20 hours. The reaction was then concentrated by ultracentrifugation after Sephadex purification and rediluted.

Protein concentration: 1.79 mg/ml

Drug/mAb ratio: 2.4.

Example 9A

Here, 5 mg of cetuximab (c = 13.6 mg/ml) in PBS was used for coupling to intermediate F9, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.05 mg/ml

Drug/mAb ratio: 2.1.

Example 9B

Here, 5 mg of anti-TWEAKR AK-1 (c = 12.2 mg/ml) in PBS was used for coupling to intermediate F9, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.84 mg/ml

Drug/mAb ratio: 2.1.

Example 10A

Here, 5 mg of cetuximab (c = 5.9 mg/ml) in PBS was used for coupling to intermediate F10, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.04 mg/ml

Drug/mAb ratio: 1.8.

Example 10B

Here, 5 mg of anti-TWEAKR AK-1 (c = 12.2 mg/ml) in PBS was used for coupling to intermediate F10, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.83 mg/ml

Drug/mAb ratio: 2.2.

Example 11A

Here, 5 mg of cetuximab (c=5.9 mg/ml) in PBS was used for coupling to intermediate F11, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.97 mg/ml

Drug/mAb ratio: 2.0.

Example 11B

Here, 5 mg of anti-TWEAKR AK-1 (c=12.2 mg/ml) in PBS were used for coupling to intermediate F11, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.86 mg/ml

Drug/mAb ratio: 2.0.

Example 12A

Here, 5 mg of cetuximab (c = 5.9 mg/ml) in PBS was used for coupling to intermediate F12, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.98 mg/ml

Drug/mAb ratio: 1.9.

Example 12B

Here, 40 mg of anti-TWEAKR AK-1 (c = 12.9 mg/ml) in PBS was used for coupling with intermediate F12, and the preparation was concentrated by ultracentrifugation after dextran gel purification, rediluted with PBS, and reconcentrated. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 9.9 mg/ml

Drug/mAb ratio: 2.5.

Example 13A

Here, 5 mg of cetuximab (c = 5.9 mg/ml) in PBS was used for coupling to intermediate F13, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.87 mg/ml

Drug/mAb ratio: 2.1.

Example 13B

Here, 5 mg of anti-TWEAKR AK-1 (c = 12.2 mg/ml) in PBS was used for coupling to intermediate F13, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.84 mg/ml

Drug/mAb ratio: 2.2.

Example 14A

Here, 5 mg of cetuximab (c = 13.2 mg/ml) in PBS was used for coupling to intermediate F14, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.32 mg/ml

Drug/mAb ratio: 3.8.

Example 14B

Here, 5 mg of anti-TWEAKR AK-1 (c = 12.2 mg/ml) in PBS was used for coupling to intermediate F14, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.23 mg/ml

Drug/mAb ratio: 2.1.

Example 14E

Here, 5 mg of trastuzumab (c = 13.4 mg/ml) in PBS was used for coupling to intermediate F14, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.21 mg/ml

Drug/mAb ratio: 2.6.

Example 15A

Here, 5 mg of cetuximab (c = 13.2 mg/ml) in PBS was used for conjugation with intermediate F15, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.39 mg/ml

Drug/mAb ratio: 3.7.

Example 15B

Here, 32 mg of anti-TWEAKR AK-1 (c = 10.1 mg/ml) in PBS was used for coupling to intermediate F15, and the preparation was concentrated by ultracentrifugation after dextran gel purification, rediluted with PBS, and reconcentrated. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 11.82 mg/ml

Drug/mAb ratio: 3.7.

Example 15E

Here, 5 mg of trastuzumab (c = 13.4 mg/ml) in PBS was used for coupling to intermediate F15, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.57 mg/ml

Drug/mAb ratio: 3.0.

Example 16A

Here, 5 mg of cetuximab (c = 13.2 mg/ml) in PBS was used for conjugation with intermediate F16, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.15 mg/ml

Drug/mAb ratio: 3.1.

Example 16B

Here, 30 mg of anti-TWEAKR AK-1 (c = 12.9 mg/ml) in PBS was used for coupling with intermediate F16, and the preparation was concentrated by ultracentrifugation after dextran gel purification, rediluted with PBS, and reconcentrated. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 9.54 mg/ml

Drug/mAb ratio: 2.8.

Example 16E

Here, 5 mg of trastuzumab (c = 13.4 mg/ml) in PBS was used for coupling to intermediate F16, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.26 mg/ml

Drug/mAb ratio: 3.4.

Example 17A

Here, 5 mg of cetuximab (c = 13.6 mg/ml) in PBS was used for coupling to intermediate F17, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 2.02 mg/ml

Drug/mAb ratio: Not available (nd).

Example 17B

Here, 5 mg of anti-TWEAKR AK-1 (c=12.2 mg/ml) in PBS were used for coupling to intermediate F17, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.64 mg/ml

Drug/mAb ratio: 2.9.

Example 18A

Here, 5 mg of cetuximab (c=13.6 mg/ml) in PBS was used for coupling to intermediate F18, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.8 mg/ml

Drug/mAb ratio: 2.7.

Example 18B

Here, 5 mg of anti-TWEAKR AK-1 (c=12.2 mg/ml) in PBS were used for coupling to intermediate F18, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.74 mg/ml

Drug/mAb ratio: 2.7.

Example 19A

Here, 5 mg of cetuximab (c = 5.9 mg/ml) in PBS was used for coupling to intermediate F19, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.94 mg/ml

Drug/mAb ratio: 2.2.

Example 19B

Here, 5 mg of anti-TWEAKR AK-1 (c = 12.2 mg/ml) in PBS was used for coupling to intermediate F19, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.9 mg/ml

Drug/mAb ratio: 2.8.

Example 20A

Here, 5 mg of cetuximab (c = 5.9 mg/ml) in PBS was used for coupling to intermediate F20, and the formulation was concentrated by ultracentrifugation after purification on a dextran gel and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.99 mg/ml

Drug/mAb ratio: 2.6.

Example 20B

Here, 5 mg of anti-TWEAKR AK-1 (c = 10.1 mg/ml) in PBS was used to couple with intermediate F20, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.03 mg/ml

Drug/mAb ratio: 2.5.

Example 21A

Here, 5 mg of cetuximab (c = 5.9 mg/ml) in PBS was used for coupling to intermediate F21, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.76 mg/ml

Drug/mAb ratio: 2.4.

Example 21B

Here, 5 mg of anti-TWEAKR AK-1 (c = 10.1 mg/ml) in PBS was used for coupling to intermediate F21, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.8 mg/ml

Drug/mAb ratio: 2.3.

Example 22A

Here, 5 mg of cetuximab (c = 13.56 mg/ml) in PBS was used for coupling to intermediate F22, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.99 mg/ml

Drug/mAb ratio: 2.9.

Example 22B

Here, 5 mg of anti-TWEAKR AK-1 (c = 12.9 mg/ml) in PBS was used for coupling to intermediate F22, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.77 mg/ml

Drug/mAb ratio: 3.0.

Example 22E

Here, 5 mg of trastuzumab (c = 13.4 mg/ml) in PBS was used for coupling to intermediate F22, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.89 mg/ml

Drug/mAb ratio: 3.0.

Example 23A

Here, 5 mg of cetuximab (c = 13.56 mg/ml) in PBS was used for coupling to intermediate F23, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted.

Protein concentration: 1.32 mg/ml

Drug/mAb ratio: 3.4.

Example 23B

Here, 5 mg of anti-TWEAKR AK-1 (c = 12.9 mg/ml) in PBS were used for coupling to intermediate F23, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.83 mg/ml

Drug/mAb ratio: 3.7.

Example 24A

Here, 5 mg of cetuximab (c = 13.56 mg/ml) in PBS was used for coupling to intermediate F24, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.83 mg/ml

Drug/mAb ratio: 2.9.

Example 24B

Here, 5 mg of anti-TWEAKR AK-1 (c = 10.1 mg/ml) in PBS was used for coupling to intermediate F24, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.83 mg/ml

Drug/mAb ratio: 2.9.

Example 25A

Here, 5 mg of cetuximab (c = 13.56 mg/ml) in PBS was used for coupling to intermediate F25, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.17 mg/ml

Drug/mAb ratio: 3.2.

Example 25B

Here, 5 mg of anti-TWEAKR AK-1 (c = 10.1 mg/ml) in PBS was used to couple with intermediate F25, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.78 mg/ml

Drug/mAb ratio: 3.2.

Example 26A

Here, 5 mg of cetuximab (c = 13.56 mg/ml) in PBS was used for conjugation with intermediate F26, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.8 mg/ml

Drug/mAb ratio: No data available.

Example 26B

Here, 5 mg of anti-TWEAKR AK-1 (c = 12.9 mg/ml) in PBS was used for coupling to intermediate F26, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.02 mg/ml

Drug/mAb ratio: 2.7.

Example 27A

Here, 5 mg of cetuximab (c = 5.9 mg/ml) in PBS was used for conjugation with intermediate F27, and the formulation was concentrated by ultracentrifugation after purification on a dextran gel and rediluted. Some of the ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.91 mg/ml

Drug/mAb ratio: 2.1.

Example 27B

Here, 5 mg of anti-TWEAKR AK-1 (c = 10.1 mg/ml) in PBS was used for coupling to intermediate F27, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.83 mg/ml

Drug/mAb ratio: 2.1.

Example 28A

Here, 5 mg of cetuximab (c = 11.59 mg/ml) in PBS was used for coupling to intermediate F28, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 2.13 mg/ml

Drug/mAb ratio: 3.6.

Example 28B

Here, 5 mg of anti-TWEAKR AK-1 (c = 10.1 mg/ml) in PBS were used for coupling to intermediate F28, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.95 mg/ml

Drug/mAb ratio: 3.5.

Example 29A

Here, 5 mg of cetuximab (c = 11.59 mg/ml) in PBS was used for coupling to intermediate F29, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 2.49 mg/ml

Drug/mAb ratio: 3.7.

Example 29B

Here, 5 mg of anti-TWEAKR AK-1 (c = 10.1 mg/ml) in PBS were used for coupling to intermediate F29, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.82 mg/ml

Drug/mAb ratio: 3.1.

Example 30A

Here, 5 mg of cetuximab (c = 11.59 mg/ml) in PBS was used for conjugation with intermediate F30, and the formulation was concentrated by ultracentrifugation after purification on a dextran gel and rediluted. Some of the ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.04 mg/ml

Drug/mAb ratio: 2.8.

Example 30B

Here, 5 mg of anti-TWEAKR AK-1 (c = 10.1 mg/ml) in PBS was used to couple with intermediate F30, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.85 mg/ml

Drug/mAb ratio: 2.9.

Example 31A

Here, 5 mg of cetuximab (c=5.1 mg/ml) in PBS was used for coupling to intermediate F31, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 2.21 mg/ml

Drug/mAb ratio: 2.1.

Example 31B

Here, 5 mg of anti-TWEAKR AK-1 (c = 16.54 mg/ml) in PBS were used for coupling to intermediate F31, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.59 mg/ml

Drug/mAb ratio: 2.4.

Example 32A

Here, 5 mg of cetuximab (c = 11.59 mg/ml) in PBS was used for conjugation with intermediate F32, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.91 mg/ml

Drug/mAb ratio: 3.6.

Example 32B

Here, 5 mg of anti-TWEAKR AK-1 (c = 10.1 mg/ml) in PBS was used for coupling to intermediate F32, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.78 mg/ml

Drug/mAb ratio: 3.6.

Example 33A

Here, 5 mg of cetuximab (c = 8.95 mg/ml) in PBS was conjugated to intermediate F33. After TCEP reduction, conjugation with the antibody was performed with stirring overnight, followed by further workup via Sephadex purification. Following Sephadex purification, the reaction was concentrated by ultracentrifugation and rediluted with PBS.

Protein concentration: 1.78 mg/ml

Drug/mAb ratio: 3.3.

Example 33B

Here, 5 mg of anti-TWEAKR AK-1 (c = 12.87 mg/ml) in PBS was used for coupling to intermediate F33. After TCEP reduction, coupling with the antibody was performed with stirring overnight, followed by further workup via Sephadex purification. Following Sephadex purification, the reaction was concentrated by ultracentrifugation and rediluted with PBS.

Protein concentration: 1.46 mg/ml

Drug/mAb ratio: 3.9.

Example 34A

Here, 5 mg of cetuximab (c = 8.95 mg/ml) in PBS was used for conjugation with intermediate F34, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.21 mg/ml

Drug/mAb ratio: 3.5.

Example 34B

Here, 5 mg of anti-TWEAKR AK-1 (c = 12.87 mg/ml) in PBS was used for coupling to intermediate F34, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.4 mg/ml

Drug/mAb ratio: 3.3.

Example 35A

Here, 5.0 mg of cetuximab (c = 5.90 mg/ml) in PBS was used for coupling to intermediate F35, and the formulation was concentrated by ultracentrifugation after purification on a dextran gel, rediluted with PBS, and reconcentrated. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 14.30 mg/ml

Drug/mAb ratio: 1.4.

Example 35B

Here, 5.0 mg of anti-TWEAKR AK-1 antibody (c = 16.54 mg/ml) in PBS was conjugated to intermediate F35, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 0.93 mg/ml

Drug/mAb ratio: 2.2.

Example 35E

Here, 5.0 mg of trastuzumab antibody (c = 8.23 mg/ml) in PBS was conjugated to intermediate F35, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.61 mg/ml

Drug/mAb ratio: 2.1.

Example 36A

Here, 5.0 mg of cetuximab (c = 5.9 mg/ml) in PBS was used for conjugation with intermediate F36, and the formulation was concentrated by ultracentrifugation after purification on a dextran gel, rediluted with PBS, and reconcentrated. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 13.86 mg/ml

Drug/mAb ratio: 2.9.

Example 36B

Here, 5.0 mg of anti-TWEAKR AK-1 antibody (c = 16.54 mg/ml) in PBS was conjugated to intermediate F36, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.08 mg/ml

Drug/mAb ratio: 2.0.

Example 36E

Here, 5.0 mg of trastuzumab antibody (c = 8.23 mg/ml) in PBS was conjugated to intermediate F36, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.98 mg/ml

Drug/mAb ratio: 1.9.

Example 37A

Here, 5.0 mg of cetuximab (c = 5.08 mg/ml) in PBS was used for conjugation with intermediate F37, and the formulation was concentrated by ultracentrifugation after purification on a dextran gel and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.52 mg/ml

Drug/mAb ratio: 1.5.

Example 37B

Here, 5.0 mg of anti-TWEAKR AK-1 antibody (c = 16.54 mg/ml) in PBS was conjugated to intermediate F37, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.91 mg/ml

Drug/mAb ratio: 2.0.

Example 37E

Here, 5.0 mg of trastuzumab antibody (c = 8.23 mg/ml) in PBS was conjugated to intermediate F37, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.23 mg/ml

Drug/mAb ratio: 1.5.

Example 38A

Here, 5.0 mg of cetuximab (c = 5.08 mg/ml) in PBS was used for conjugation with intermediate F38, and the formulation was concentrated by ultracentrifugation after purification on a dextran gel and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.45 mg/ml

Drug/mAb ratio: 1.9.

Example 38B

Here, 5.0 mg of anti-TWEAKR AK-1 antibody (c = 12.23 mg/ml) in PBS was conjugated to intermediate F38, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.54 mg/ml

Drug/mAb ratio: 2.2.

Example 38E

Here, 5.0 mg of trastuzumab antibody (c = 13.39 mg/ml) in PBS was conjugated to intermediate F38, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.90 mg/ml

Drug/mAb ratio: 2.4.

Example 39A

Here, 5.0 mg of cetuximab (c = 5.90 mg/ml) in PBS was used for conjugation with intermediate F39, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.98 mg/ml

Drug/mAb ratio: 2.0.

Example 39B

Here, 5.0 mg of anti-TWEAKR AK-1 antibody (c = 12.23 mg/ml) in PBS was conjugated to intermediate F39, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.40 mg/ml

Drug/mAb ratio: 1.4.

Example 39E

Here, 5.0 mg of trastuzumab antibody (c = 13.39 mg/ml) in PBS was conjugated to intermediate F39, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.04 mg/ml

Drug/mAb ratio: 2.1.

Example 40A

Here, 5.0 mg of cetuximab (c = 5.90 mg/ml) in PBS was used for conjugation with intermediate F40, and the formulation was concentrated by ultracentrifugation after purification on a dextran gel and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.0 mg/ml

Drug/mAb ratio: 2.5.

Example 40B

Here, 5.0 mg of anti-TWEAKR AK-1 antibody (c = 12.23 mg/ml) in PBS was conjugated to intermediate F40, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.81 mg/ml

Drug/mAb ratio: 2.5.

Example 40E

Here, 5.0 mg of trastuzumab antibody (c = 13.39 mg/ml) in PBS was conjugated to intermediate F40, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.98 mg/ml

Drug/mAb ratio: 2.3.

Example 41A

Here, 5.0 mg of cetuximab (c = 5.08 mg/ml) in PBS was used for conjugation with intermediate F41, and the formulation was concentrated by ultracentrifugation after purification on a dextran gel and rediluted. Some of the ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.53 mg/ml

Drug/mAb ratio: 1.9.

Example 41B

Here, 5.0 mg of anti-TWEAKR AK-1 antibody (c = 12.23 mg/ml) in PBS was conjugated to intermediate F41, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.73 mg/ml

Drug/mAb ratio: 2.5.

Example 41E

Here, 5.0 mg of trastuzumab antibody (c = 13.39 mg/ml) in PBS was conjugated to intermediate F39, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.09 mg/ml

Drug/mAb ratio: 2.5.

Example 42A

Here, 5.0 mg of cetuximab (c = 5.90 mg/ml) in PBS was used for conjugation with intermediate F42, and the formulation was concentrated by ultracentrifugation after purification on a dextran gel and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.05 mg/ml

Drug/mAb ratio: 2.1.

Example 42B

Here, 5.0 mg of anti-TWEAKR AK-1 antibody (c = 12.23 mg/ml) in PBS was conjugated to intermediate F42, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.64 mg/ml

Drug/mAb ratio: 2.3.

Example 42E

Here, 5.0 mg of trastuzumab antibody (c = 13.39 mg/ml) in PBS was conjugated to intermediate F42, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.80 mg/ml

Drug/mAb ratio: 2.0.

Example 43A

Here, 5.0 mg of cetuximab (c = 5.90 mg/ml) in PBS was used for conjugation with intermediate F43, and the formulation was concentrated by ultracentrifugation after purification on a dextran gel and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.90 mg/ml

Drug/mAb ratio: 1.3.

Example 43B

Here, 5.0 mg of anti-TWEAKR AK-1 antibody (c = 12.23 mg/ml) in PBS was conjugated to intermediate F43, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 0.85 mg/ml

Drug/mAb ratio: 1.3.

Example 43E

Here, 5.0 mg of trastuzumab antibody (c = 13.39 mg/ml) in PBS was conjugated to intermediate F43, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.97 mg/ml

Drug/mAb ratio: 2.1.

Example 44A

Here, 5.0 mg of cetuximab (c = 5.90 mg/ml) in PBS was used for conjugation with intermediate F44, and the formulation was concentrated by ultracentrifugation after purification on a dextran gel and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.99 mg/ml

Drug/mAb ratio: 2.5.

Example 44B

Here, 5.0 mg of anti-TWEAKR AK-1 antibody (c = 12.23 mg/ml) in PBS was conjugated to intermediate F44, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.65 mg/ml

Drug/mAb ratio: 2.5.

Example 44E

Here, 5.0 mg of trastuzumab antibody (c = 13.39 mg/ml) in PBS was conjugated to intermediate F44, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.95 mg/ml

Drug/mAb ratio: 2.4.

Example 45A

Here, 5.0 mg of cetuximab (c = 5.90 mg/ml) in PBS was used for conjugation with intermediate F45, and the formulation was concentrated by ultracentrifugation after purification on a dextran gel and rediluted. Some of the ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.92 mg/ml

Drug/mAb ratio: 2.3.

Example 45B

Here, 5.0 mg of anti-TWEAKR AK-1 antibody (c = 12.23 mg/ml) in PBS was conjugated to intermediate F45, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.27 mg/ml

Drug/mAb ratio: 1.8.

Example 45E

Here, 5.0 mg of trastuzumab antibody (c = 13.39 mg/ml) in PBS was conjugated to intermediate F45, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.91 mg/ml

Drug/mAb ratio: 2.1.

Example 46A

Here, 5.0 mg of cetuximab (c = 5.90 mg/ml) in PBS was used for conjugation with intermediate F46, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.0 mg/ml

Drug/mAb ratio: 2.2.

Example 46B

Here, 5.0 mg of anti-TWEAKR AK-1 antibody (c = 12.23 mg/ml) in PBS was conjugated to intermediate F46, and the formulation was concentrated by ultracentrifugation after dextran gel purification, diluted, and reconcentrated. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 11.21 mg/ml

Drug/mAb ratio: 2.1.

Example 46E

Here, 5.0 mg of trastuzumab antibody (c = 13.39 mg/ml) in PBS was conjugated to intermediate F46, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.84 mg/ml

Drug/mAb ratio: 2.4.

Example 47A

Here, 5.0 mg of cetuximab (c = 5.90 mg/ml) in PBS was used for conjugation with intermediate F47, and the formulation was concentrated by ultracentrifugation after purification on a dextran gel and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.51 mg/ml

Drug/mAb ratio: 2.1.

Example 47B

Here, 5.0 mg of anti-TWEAKR AK-1 antibody (c = 12.23 mg/ml) in PBS was conjugated to intermediate F47, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 0.89 mg/ml

Drug/mAb ratio: 1.7.

Example 47E

Here, 5.0 mg of trastuzumab antibody (c = 13.39 mg/ml) in PBS was conjugated to intermediate F47, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.97 mg/ml

Drug/mAb ratio: 2.9.

Example 48A

Here, 5.0 mg of cetuximab (c = 5.90 mg/ml) in PBS was used for conjugation with intermediate F48, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.10 mg/ml

Drug/mAb ratio: 2.4.

Example 48B

Here, 5.0 mg of anti-TWEAKR AK-1 antibody (c = 10.10 mg/ml) in PBS was conjugated to intermediate F48, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.73 mg/ml

Drug/mAb ratio: 1.8.

Example 48E

Here, 5.0 mg of trastuzumab antibody (c = 13.39 mg/ml) in PBS was conjugated to intermediate F48, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.95 mg/ml

Drug/mAb ratio: 2.7.

Example 49A

Here, 5.0 mg of cetuximab (c = 5.90 mg/ml) in PBS was used for conjugation with intermediate F49, and the formulation was concentrated by ultracentrifugation after purification on a dextran gel and rediluted. Some of the ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.09 mg/ml

Drug/mAb ratio: 2.0.

Example 49B

Here, 5.0 mg of anti-TWEAKR AK-1 antibody (c = 12.87 mg/ml) in PBS was conjugated to intermediate F49, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.95 mg/ml

Drug/mAb ratio: 2.1.

Example 49E

Here, 5.0 mg of trastuzumab antibody (c = 13.5 mg/ml) in PBS was conjugated to intermediate F49, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.96 mg/ml

Drug/mAb ratio: 2.4.

Example 50A

Here, 5 mg of cetuximab (c = 11.02 mg/ml) in PBS was used for coupling to intermediate F50, and the formulation was concentrated by ultracentrifugation after purification on a dextran gel and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.89 mg/ml

Drug/mAb ratio: 3.0.

Example 50B

Here, 5 mg of anti-TWEAKR AK-1 (c = 12.87 mg/ml) in PBS was used for coupling to intermediate F50, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.82 mg/ml

Drug/mAb ratio: 2.9.

Example 51A

Here, 5 mg of cetuximab (c = 5.1 mg/ml) in PBS was conjugated to the intermediate 3-{3-[(3-amino-2-{[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]methyl}propyl)sulfanyl]-2,5-dioxopyrrolidin-1-yl}-N-[17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-15-oxo-4,7,10-trioxa-14-azaheptadecan-1-yl]propanamide (isomer 1), and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist as hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.16 mg/ml.

Example 51B

Here, 5 mg of anti-TWEAKR AK-1 (c = 16.5 mg/ml) in PBS was conjugated to the intermediate 3-{3-[(3-amino-2-{[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]methyl}propyl)sulfanyl]-2,5-dioxopyrrolidin-1-yl}-N-[17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-15-oxo-4,7,10-trioxa-14-azaheptadecan-1-yl]propanamide (isomer 1), and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist as hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.74 mg/ml

Drug/mAb ratio: 1.7.

Example 51E

Here, 5 mg of trastuzumab (c = 8.2 mg/ml) in PBS was used to couple with the intermediate 3-{3-[(3-amino-2-{[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]methyl}propyl)sulfanyl]-2,5-dioxopyrrolidin-1-yl}-N-[17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-15-oxo-4,7,10-trioxa-14-azaheptadecan-1-yl]propanamide (isomer 1), and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.67 mg/ml

Drug/mAb ratio: 2.4.

Example 52A

Here, 5 mg of cetuximab (c = 5.1 mg/ml) in PBS was conjugated to the intermediate 3-{3-[(3-amino-2-{[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]methyl}propyl)sulfanyl]-2,5-dioxopyrrolidin-1-yl}-N-[17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-15-oxo-4,7,10-trioxa-14-azaheptadecan-1-yl]propanamide (isomer 2), and the formulation was concentrated by ultracentrifugation after purification on a dextran gel and rediluted with PBS. Some ADC may also exist as hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.41 mg/ml

Drug/mAb ratio: 2.3.

Example 52B

Here, 5 mg of anti-TWEAKR AK-1 (c = 16.5 mg/ml) in PBS was used to couple with the intermediate 3-{3-[(3-amino-2-{[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]methyl}propyl)sulfanyl]-2,5-dioxopyrrolidin-1-yl}-N-[17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-15-oxo-4,7,10-trioxa-14-azaheptadecan-1-yl]propanamide (isomer 2), and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.34 mg/ml

Drug/mAb ratio: 1.6.

Example 52E

Here, 5 mg of trastuzumab (c = 8.2 mg/ml) in PBS was used to couple with the intermediate 3-{3-[(3-amino-2-{[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]methyl}propyl)sulfanyl]-2,5-dioxopyrrolidin-1-yl}-N-[17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-15-oxo-4,7,10-trioxa-14-azaheptadecan-1-yl]propanamide (isomer 2), and the formulation was concentrated by ultracentrifugation after purification on a Sephadex gel and rediluted with PBS. Some ADC may also exist as hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.16 mg/ml

Drug/mAb ratio: 2.6.

Example 53A

Here, 5 mg of cetuximab (c = 5.1 mg/ml) in PBS was used for coupling with the intermediate N-{3-amino-2-[({1-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl]-2,5-dioxopyrrolidin-3-yl}sulfanyl)methyl]propyl}-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide (isomer 1), and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 2.09 mg/ml

Drug/mAb ratio: 2.1.

Example 53B

Here, 5 mg of anti-TWEAKR AK-1 (c = 16.5 mg/ml) in PBS were used for coupling with the intermediate N-{3-amino-2-[({1-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl]-2,5-dioxopyrrolidin-3-yl}sulfanyl)methyl]propyl}-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide (isomer 1), and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.95 mg/ml

Drug/mAb ratio: 1.8.

Example 53E

Here, 5 mg of trastuzumab (c = 8.2 mg/ml) in PBS was used for coupling with the intermediate N-{3-amino-2-[({1-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl]-2,5-dioxopyrrolidin-3-yl}sulfanyl)methyl]propyl}-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide (isomer 1), and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 2.15 mg/ml

Drug/mAb ratio: 2.4.

Example 54A

Here, 5 mg of cetuximab (c = 5.1 mg/ml) in PBS was used for coupling with the intermediate N-{3-amino-2-[({1-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl]-2,5-dioxopyrrolidin-3-yl}sulfanyl)methyl]propyl}-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide (isomer 2), and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 2.17 mg/ml

Drug/mAb ratio: 1.9.

Example 54B

Here, 5 mg of anti-TWEAKR AK-1 (c = 16.5 mg/ml) in PBS was used for coupling with the intermediate N-{3-amino-2-[({1-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl]-2,5-dioxopyrrolidin-3-yl}sulfanyl)methyl]propyl}-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide (isomer 2), and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.95 mg/ml

Drug/mAb ratio: 2.0.

Example 54E

Here, 5 mg of trastuzumab (c = 8.2 mg/ml) in PBS was used for coupling with the intermediate N-{3-amino-2-[({1-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl]-2,5-dioxopyrrolidin-3-yl}sulfanyl)methyl]propyl}-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide (isomer 2), and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.90 mg/ml

Drug/mAb ratio: 1.8.

Example 55A

Here, 5 mg of cetuximab (c = 5.1 mg/ml) in PBS was used to couple with the intermediate N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecanoyl]-L-valyl-N-{4-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}carbamoyl]phenyl}-L-alaninamide, and the formulation was concentrated by ultracentrifugation after purification on a dextran gel and rediluted with PBS. Some ADC may also exist as hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 0.47 mg/ml

Drug/mAb ratio: No data available.

Example 55B

Here, 5 mg of anti-TWEAKR AK-1 (c = 12.2 mg/ml) in PBS was used to couple with the intermediate N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecanoyl]-L-valyl-N-{4-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}carbamoyl]phenyl}-L-alaninamide, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.26 mg/ml

Drug/mAb ratio: 1.8.

Example 56A

Here, 5 mg of cetuximab (c = 15.33 mg/ml) in PBS was used for conjugation with intermediate F56, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.74 mg/ml

Drug/mAb ratio: 2.8.

Example 56B

Here, 5 mg of anti-TWEAKR AK-1 (c = 12.87 mg/ml) in PBS was used for coupling to intermediate F56, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.92 mg/ml

Drug/mAb ratio: 2.8.

Example 57A

Here, 5 mg of cetuximab (c = 15.33 mg/ml) in PBS was used for conjugation with intermediate F57, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.04 mg/ml

Drug/mAb ratio: 3.3.

Example 57B

Here, 5 mg of anti-TWEAKR AK-1 (c = 12.87 mg/ml) in PBS was used for coupling to intermediate F57, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.93 mg/ml

Drug/mAb ratio: 3.3.

Example 58A

Here, 20 mg of cetuximab (c = 21.32 mg/ml) in PBS was used for coupling to intermediate F58, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.33 mg/ml

Drug/mAb ratio: 2.0.

Example 58B

Here, 20.0 mg of anti-TWEAKR AK-1 antibody (c=18.6 mg/ml) in PBS was used for coupling to intermediate F58, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.54 mg/ml

Drug/mAb ratio: 4.6.

Example 58E

Here, 5.0 mg of trastuzumab antibody (c = 13.5 mg/ml) in PBS was used for coupling to intermediate F58, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.15 mg/ml.

Example 59

N ⁶ -{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butyryl}-L-lysine trifluoroacetic acid (1:1)

8 mg (15 μmol) of intermediate F1 was placed in 1.7 ml of DCM and 7.3 mg (30 μmol) of N2-(tert-butoxycarbonyl)-L-lysine and 13 μl of N , N -diisopropylethylamine were added. The reaction mixture was stirred at room temperature for 15 minutes, then concentrated under vacuum and purified by preparative HPLC. The intermediate product was then placed in 1 ml of DCM and deprotected using 1 ml of TFA. The preparation was concentrated and the residue was lyophilized from acetonitrile/water 1:1.

LC-MS (Method 1): R _t = 0.8 min; MS (ESIpos): m/z = 643 (M+H) ⁺ .

Example 60

S-{1-[6-(2-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}hydrazino)-6-oxohexyl]-2,5-dioxopyrrolidin-3-yl}-L-cysteine

4 mg (5 μmol) of intermediate F3 were dissolved in 1 ml of DCM and 1.2 mg (10 μmol) of L-cysteine were added. The reaction mixture was stirred at room temperature for 20 h, then concentrated under vacuum and purified by preparative HPLC.

LC-MS (Method 1): R _t = 0.85 min; MS (ESIpos): m/z = 843 (M+H) ⁺ .

Example 61

S-{1-[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]-2,5-dioxopyrrolidin-3-yl}-L-cysteine

7 mg (9 μmol) of intermediate F2 were placed in 1.75 mL of DMF and 2.3 mg (19 μmol) of L-cysteine were added. The reaction mixture was stirred at room temperature for 20 h, then concentrated under vacuum and purified by preparative HPLC.

LC-MS (Method 1): R _t = 0.84 min; MS (ESIpos): m/z = 758 (M+H) ⁺ .

Example 62

S-(1-{2-[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(propionyl)amino]butyryl}amino)ethoxy]ethyl}-2,5-dioxopyrrolidin-3-yl)-L-cysteine

2.9 mg (3.6 μmol) of Intermediate F5 was placed in 1 mL of DMF and 0.9 mg (7.3 μmol) of L-cysteine was added. The reaction mixture was stirred at room temperature for 3 days, after which the same amount of cysteine was added. After stirring at room temperature for an additional 24 hours, the mixture was concentrated under vacuum, and the product was purified by preparative HPLC.

LC-MS (Method 1): R _t = 0.84 min; MS (ESIpos): m/z = 802 (M+H) ⁺ .

Example 63

N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexanoyl]-L-valyl-L-alanyl-N ⁶ -{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butyryl}-L-lysine trifluoroacetic acid (1:1)

3 mg (3 μmol) of Intermediate F8 was dissolved in 2 mL of DMF and 1 mg (8 μmol) of L-cysteine was added. The reaction mixture was stirred at room temperature for 10 minutes, 500 μl of water was added, and the mixture was adjusted to pH 2 using TFA, then concentrated under vacuum and purified by preparative HPLC.

LC-MS (Method 1): R _t = 0.82 min; MS (ESIpos): m/z = 1127 (M+H) ⁺ .

Example 64

S-[1-(2-{[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]amino}-2-oxoethyl)-2,5-dioxopyrrolidin-3-yl]-L-cysteine trifluoroacetic acid (1:1)

5.6 mg (7 μmol) of intermediate F7 were dissolved in 1 ml of DMF and 1.7 mg (14 μmol) of L-cysteine were added. The reaction mixture was stirred at room temperature for 20 hours and then concentrated under vacuum. The product was purified by preparative HPLC.

LC-MS (Method 1): R _t = 0.81 min; MS (ESIpos): m/z = 815 (M+H) ⁺ .

Example 65

N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl-L-alanyl-N-(4-{[(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)acetyl]amino}phenyl)-N ⁵ -carbamoyl-L-ornithinamide

3 mg (3 μmol) of intermediate F25 was dissolved in 2 mL of DMF and 200 μL of water, and 1 mg (8 μmol) of L-cysteine was added. The reaction mixture was stirred at room temperature for 4 hours, then 500 μL of water was added and the pH was adjusted to 2 using TFA, then concentrated under vacuum and purified by preparative HPLC.

LC-MS (Method 1): R _t = 0.76 min; MS (ESIpos): m/z = 1162 (M+H) ⁺ .

Example 66

S-(1-{4-[(N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl)amino]phenyl}-2,5-dioxopyrrolidin-3-yl)-L-cysteine

3.5 mg (3.8 μmol) of Intermediate F10 was dissolved in 1 mL of DMF and 1.4 mg (11 μmol) of L-cysteine was added. The reaction mixture was stirred at room temperature for 23 hours, after which the same amount of cysteine was added. After stirring at room temperature for an additional 24 hours, the mixture was concentrated under vacuum, and the product was purified by preparative HPLC.

LC-MS (Method 1): R _t = 0.82 min; MS (ESIpos): m/z = 877 (M+H) ⁺ .

Example 67

S-{1-[2-({[(1R,2S)-2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)cyclopentyl]carbonyl}amino)ethyl]-2,5-dioxopyrrolidin-3-yl}-L-cysteine

3.3 mg (4 μmol) of Intermediate F12 was dissolved in 1 mL of DMF and 1.4 mg (11 μmol) of L-cysteine was added. The reaction mixture was stirred at room temperature for 3 hours. 500 μL of water was then added, and the mixture was concentrated under vacuum. The product was purified by preparative HPLC.

LC-MS (Method 1): R _t = 0.82 min; MS (ESIpos): m/z = 869 (M+H) ⁺ .

Example 68

N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl-L-alanyl-N-[4-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)phenyl]-N ⁵ -carbamoyl-L-ornithinamide

3 mg (3 μmol) of Intermediate F15 was dissolved in 2 mL of DMF and 1 mg (8 μmol) of L-cysteine was added. The reaction mixture was stirred at room temperature for 4 hours. 500 μL of water was then added to the mixture, and the pH was adjusted to 2 using TFA. The mixture was then concentrated under vacuum, and the product was purified by preparative HPLC.

LC-MS (Method 1): R _t = 0.76 min; MS (ESIpos): m/z = 1105 (M+H) ⁺ .

Example 69

N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl-L-valyl-N-[4-(2-{[2-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)ethyl]amino}-2-oxoethyl)phenyl]-N ⁵ -carbamoyl-L-ornithinamide

3 mg (2 μmol) of Intermediate F16 was placed in 2 mL of DMF and 0.8 mg (7 μmol) of L-cysteine was added. The reaction mixture was stirred at room temperature for 4 hours. 500 μL of water was then added to the mixture, and the pH was adjusted to 2 using TFA. The mixture was then concentrated under vacuum, and the product was purified by preparative HPLC.

LC-MS (Method 1): R _t = 0.82 min; MS (ESIpos): m/z = 1218 (M+H) ⁺ .

Example 70

N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl-L-alanyl-N-[4-(2-{[2-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)ethyl]amino}-2-oxoethyl)phenyl]-N ⁵ -carbamoyl-L-ornithinamide

3 mg (3 μmol) of Intermediate F24 was dissolved in 2 mL of DMF and 200 μL of water, and 0.9 mg (8 μmol) of L-cysteine was added. The reaction mixture was stirred at room temperature for 4 hours. 500 μL of water was then added to the mixture, and the pH was adjusted to 2 using TFA. The mixture was then concentrated under vacuum, and the product was purified by preparative HPLC.

LC-MS (Method 1): R _t = 0.77 min; MS (ESIpos): m/z = 1190 (M+H) ⁺ .

Example 71

(15S,19R)-15-amino-19-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-18-glycoloyl-20,20-dimethyl-14-oxo-4,7,10-trioxa-13,18-diazaheneicosane-1-oic acid/trifluoroacetic acid (1:1)

In the first step, 70 mg (0.114 mmol) of intermediate C5 were coupled with 32 mg (0.114 mmol) of tert-butyl 3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propanoate in 15 ml of DMF in the presence of 44 mg (0.228 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 35 mg (0.228 mmol) of 1-hydroxy- 1H -benzotriazole hydrate and 60 μl of N , N -diisopropylethylamine.

The preparation was stirred at room temperature overnight and the product was purified by preparative HPLC. This yielded 33 mg (33% of theory) of the protected intermediate. This was stirred with 1.1 ml of trifluoroacetic acid in 11 ml of dichloromethane for 1 hour, yielding, after workup, 26 mg (98%) of the title compound.

LC-MS (Method 1): R _t = 0.91 min; MS (ESIpos): m/z = 718 (M+H) ⁺ .

Example 72

N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl-L-valyl-N ⁵ -carbamoyl-L-ornithine/trifluoroacetic acid (1:1)

The title compound was prepared from intermediate C3 using classical methods of peptide chemistry.

LC-MS (Method 1): R _t = 0.87 min; MS (ESIpos): m/z = 842 (M+H) ⁺ .

Example 73

N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl-L-alanyl-N ⁵ -carbamoyl-L-ornithine/trifluoroacetic acid (1:1)

The title compound was prepared analogously to Example 72 from intermediate C3 using classical methods of peptide chemistry.

LC-MS (Method 1): R _t = 0.82 min; MS (ESIpos): m/z = 814 (M+H) ⁺ .

Example 74

N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl-4-amino-L-phenylalanine/trifluoroacetic acid (1:2)

The title compound was synthesized from intermediate C8 and trifluoroacetic acid/4-amino-L-phenylalanine methyl ester (1:1) prepared from commercially available N-(tert-butoxycarbonyl)-4-nitro-L-phenylalanine using classical methods of peptide chemistry.

LC-MS (Method 1): R _t = 0.82 min; MS (ESIpos): m/z = 748 (M+H) ⁺ .

Example 75

Trifluoroacetic acid/N-{(1R)-1-[1-(3-aminobenzyl)-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-N-(3-aminopropyl)-2-hydroxyacetamide (1:1)

The title compound was prepared from intermediate C10 by deprotection with trifluoroacetic acid.

LC-MS (Method 1): R _t = 0.81 min; MS (ESIpos): m/z = 486 (M+H) ⁺ .

Example 76

(2S)-2-Amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butanoic acid/trifluoroacetic acid (1:1)

The title compound was prepared from intermediate C5 by deprotection with trifluoroacetic acid.

LC-MS (Method 1): R _t = 0.93 min; MS (ESIpos): m/z = 515 (M+H) ⁺ .

Example 77

Trifluoroacetic acid/N-[(3S)-3-amino-4-hydrazino-4-oxobutyl]-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide (1:1)

The title compound was prepared from intermediate C6 by deprotection with trifluoroacetic acid.

LC-MS (Method 1): R _t = 0.88 min; MS (ESIpos): m/z = 529 (M+H) ⁺ .

Example 78

Trifluoroacetic acid/N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-N-[(3S)-3,4-diaminobutyl]-2-hydroxyacetamide (1:1)

In the first step, intermediate C1 is reacted with benzyl tert-butyl [(2S)-4-oxobutane-1,2-diyl]biscarbamate in a similar manner to intermediate C2. The aldehyde used is prepared in a similar manner to intermediate C2 from commercially available (3S)-3-{[(benzyloxy)carbonyl]amino}-4-[(tert-butoxycarbonyl)amino]butanoic acid by reduction and subsequent oxidation.

In the second step, N-acylation was carried out analogously to intermediate C3 and finally deprotected completely first with 33% hydrobromic acid solution in glacial acetic acid and then with lithium hydroxide.

LC-MS (Method 1): R _t = 0.78 min; MS (ESIpos): m/z = 500 (M+H) ⁺ .

Example 79

N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butyryl}-β-alanine

The title compound was prepared from intermediate C8 by deprotection with trifluoroacetic acid.

LC-MS (Method 1): R _t = 0.84 min; MS (ESIpos): m/z = 586 (M+H) ⁺ .

Example 80

Trifluoroacetic acid/(2S)-2-amino-N-(2-aminoethyl)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butanamide (2:1)

The title compound was prepared from intermediate C5 using classical methods of peptide chemistry.

LC-MS (Method 1): R _t = 0.72 min; MS (ESIpos): m/z = 557 (M+H) ⁺ .

Example 81

(1-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}hydrazino)acetic acid/trifluoroacetic acid (1:2)

The title compound was prepared from intermediate C7 by deprotection with trifluoroacetic acid.

LC-MS (Method 1): R _t = 0.89 min; MS (ESIpos): m/z = 587 (M+H) ⁺ .

Example 82A

Here, 5.0 mg of cetuximab (c = 5.90 mg/ml) in PBS was used for conjugation with intermediate F82, and the formulation was concentrated by ultracentrifugation after purification on a dextran gel and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.59 mg/ml

Drug/mAb ratio: 2.3.

Example 82B

Here, 5.0 mg of anti-TWEAKR AK-1 antibody (c = 10.10 mg/ml) in PBS was conjugated to intermediate F82, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.59 mg/ml

Drug/mAb ratio: 1.8.

Example 82E

Here, 5.0 mg of trastuzumab antibody (c = 11.50 mg/ml) in PBS was conjugated to intermediate F82, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.15 mg/ml

Drug/mAb ratio: 2.7.

Example 83A

Here, 5.0 mg of cetuximab (c = 5.90 mg/ml) in PBS was used for conjugation with intermediate F83, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.77 mg/ml

Drug/mAb ratio: 2.0.

Example 83B

Here, 5.0 mg of anti-TWEAKR AK-1 antibody (c = 12.87 mg/ml) in PBS was conjugated to intermediate F83, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.29 mg/ml

Drug/mAb ratio: 1.3.

Example 83E

Here, 5.0 mg of trastuzumab antibody (c = 13.50 mg/ml) in PBS was conjugated to intermediate F83, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.91 mg/ml

Drug/mAb ratio: 2.1.

Example 83H

Here, 5.0 mg of panitumumab (c = 10 mg/ml) in PBS was coupled to intermediate F83. The reduction time with TCEP was increased to 4 hours, and the stirring time for ADC coupling was increased to 20 hours. The formulation was then concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also be present as hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.57 mg/ml

Drug/mAb ratio: 0.9.

Example 84A

Here, 5 mg of cetuximab (c = 11.02 mg/ml) in PBS was used for coupling to intermediate F84, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.94 mg/ml

Drug/mAb ratio: 2.9.

Example 84B

Here, 5 mg of anti-TWEAKR AK-1 (c = 12.9 mg/ml) in PBS was used for coupling to intermediate F84, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.77 mg/ml

Drug/mAb ratio: 3.0.

Example 85A

Here, 5 mg of cetuximab (c = 11.02 mg/ml) in PBS was used for conjugation with intermediate F85, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.13 mg/ml

Drug/mAb ratio: 3.4.

Example 85B

Here, 5 mg of anti-TWEAKR AK-1 (c = 12.9 mg/ml) in PBS was used to couple with intermediate F85, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.63 mg/ml

Drug/mAb ratio: 3.2.

Example 86A

Here, 5 mg of cetuximab (c = 11.59 mg/ml) in PBS was used for conjugation with intermediate F86, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.96 mg/ml

Drug/mAb ratio: 3.2.

Example 86B

Here, 5 mg of anti-TWEAKR AK-1 (c = 12.9 mg/ml) in PBS was used to couple with intermediate F86, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.55 mg/ml

Drug/mAb ratio: 2.8.

Example 87A

Here, 5 mg of cetuximab (c = 8.95 mg/ml) in PBS was used for conjugation with intermediate F87, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.08 mg/ml

Drug/mAb ratio: 3.5.

Example 87B

Here, 5 mg of anti-TWEAKR AK-1 (c = 12.9 mg/ml) in PBS was used to couple with intermediate F87, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.93 mg/ml

Drug/mAb ratio: 2.2.

Example 88A

Here, 5 mg of cetuximab (c = 11.02 mg/ml) in PBS was used for coupling to intermediate F88, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 2.03 mg/ml

Drug/mAb ratio: 3.1.

Example 88B

Here, 5 mg of anti-TWEAKR AK-1 (c=12.9 mg/ml) in PBS were used for coupling to intermediate F88, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.9 mg/ml

Drug/mAb ratio: 3.3.

Example 89A

Here, 5 mg of cetuximab (c = 11.02 mg/ml) in PBS was used for conjugation with intermediate F89, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.2 mg/ml

Drug/mAb ratio: 3.3.

Example 89B

Here, 5 mg of anti-TWEAKR AK-1 (c = 12.9 mg/ml) in PBS was used to couple with intermediate F89, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.03 mg/ml

Drug/mAb ratio: 3.4.

Example 90A

Here, 5 mg of cetuximab (c = 13.33 mg/ml) in PBS was used for coupling to intermediate F90, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted.

Protein concentration: 2.19 mg/ml

Drug/mAb ratio: 3.0.

Example 90B

Here, 5 mg of anti-TWEAKR AK-1 (c = 12.9 mg/ml) in PBS were used for coupling to intermediate F90, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.97 mg/ml

Drug/mAb ratio: 2.9.

Example 91A

Here, 80 mg of cetuximab (c = 5.9 mg/ml) in PBS was used for coupling to intermediate F91, and the formulation was concentrated by ultracentrifugation after purification on a dextran gel, rediluted with PBS, and reconcentrated. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 12.75 mg/ml

Drug/mAb ratio: 3.7.

Example 91B

Here, 5 mg of anti-TWEAKR AK-1 (c = 12.9 mg/ml) in PBS was used to couple with intermediate F91, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 5.71 mg/ml

Drug/mAb ratio: 4.0.

Example 92

S-(1-{2-[(N-{(2S)-2-amino-4-[{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl)amino]ethyl}-2,5-dioxopyrrolidin-3-yl)-L-cysteine trifluoroacetic acid (1:1)

3 mg (4 μmol) of intermediate F86 were dissolved in 3 ml of DCM/water 10:1 and 1.3 mg (11 μmol) of L-cysteine were added. The reaction mixture was stirred at room temperature for 10 minutes, then concentrated under vacuum and purified by preparative HPLC.

LC-MS (Method 1): R _t = 0.77 min; MS (EIpos): m/z = 829 [M+H] ⁺ .

Example 93

N-[3-Amino-2-(sulfanylmethyl)propyl]-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide hydrochloride (1:1) (Isomer 1)

This compound was obtained according to Intermediate C32 (Isomer 1).

Example 94

Intermediate C33 (Isomer 2) N-[3-Amino-2-(sulfanylmethyl)propyl]-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide hydrochloride (1:1) (Isomer 2)

This compound was obtained according to intermediate C33 (isomer 2).

Example 95

Trifluoroacetic acid/4-amino-N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}benzamide (2:1)

The title compound was obtained from tert-butyl {3-[(4-aminobenzoyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino]propyl}carbamate by deprotection with trifluoroacetic acid.

LC-MS (Method 1): R _t = 0.93 min; MS (EIpos): m/z = 532 [M+H] ⁺ .

Example 96

N-(3-Aminopropyl)-N-{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide

101 mg (0.16 mmol) of 2-({(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}[3-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)propyl]amino)-2-oxoethyl acetate were initially charged in 2 ml of pure ethanol, and 244 mg (3.14 mmol, 225 μl) of 40% aqueous methylamine solution were added. After stirring at 50° C. for 1 hour, a further 244 mg (3.14 mmol, 225 μl) of 40% aqueous methylamine solution were then added and, after a total of 3.5 hours, the mixture was purified directly by preparative HPLC (eluent: ACN/water + 1.0% NEt ₃ , gradient). This gave 52 mg (70% of theory) of the target compound.

LC-MS (Method 3): R _t = 2.56 min; MS (EIpos): m/z = 471 [M+H] ⁺ .

Example 97

S-(2-{[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]amino}-2-oxoethyl)-L-cysteine/trifluoroacetic acid (1:1)

3 mg (4 μmol) of intermediate F33 were dissolved in 2 ml of DMF and 200 μl of water, and 1.4 mg (12 μmol) of L-cysteine was added. The reaction mixture was stirred at room temperature for 1 hour. The preparation was then concentrated under vacuum, and the product was purified by preparative HPLC.

LC-MS (Method 1): R _t = 0.81 min; MS (ESIpos): m/z = 718 (M+H) ⁺ .

Example 98

N-(3-Aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide

Initially, 150.0 mg (0.42 mmol) of (1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropan-1-amine (Intermediate C52) was charged in 2.0 mL of dichloromethane. 29.2 mg (0.49 mmol) of HOAc and 125.6 mg (0.59 mmol) of sodium triacetoxyborohydride were added, and the mixture was stirred at room temperature for 5 minutes. 98.9 mg (0.49 mmol) of 3-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)propanal were added. The reaction mixture was stirred at room temperature overnight. The reaction mixture was then diluted with ethyl acetate, and the organic phase was washed twice with saturated sodium carbonate solution and once with saturated NaCl solution. After drying over magnesium sulfate, the solvent was evaporated under vacuum, and the residue was purified on silica gel (eluent: dichloromethane/methanol 100:1). The solvent was evaporated under vacuum and the residue was dried under high vacuum. This yielded 188.6 mg (74%) of the compound 2-[3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)propyl]-1H-isoindole-1,3(2H)-dione.

LC-MS (Method 1): R _t = 1.00 min; MS (ESIpos): m/z = 541 [M+H] ⁺ .

171.2 mg (0.32 mmol) of 2-[3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)propyl]-1H-isoindole-1,3(2H)-dione was initially charged in 5.0 ml of dichloromethane, and 73.6 mg (0.73 mmol) of triethylamine was added. 94.9 mg (0.70 mmol) of acetoxyacetyl chloride was added at 0°C, and the reaction mixture was stirred overnight at room temperature. The reaction mixture was diluted with ethyl acetate, and the organic phase was washed twice with saturated sodium bicarbonate solution and once with saturated NaCl solution. After drying over magnesium sulfate, the solvent was evaporated under vacuum, and the residue was purified using Biotage Isolera (silica gel, column 10 g SNAP, flow rate 12 ml/min, ethyl acetate/cyclohexane 1:3). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This resulted in 159.0 mg (77%) of the compound 2-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}[3-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)propyl]amino)-2-oxoethyl acetate.

LC-MS (Method 1): R _t = 1.35 min; MS (ESIpos): m/z = 642 [M+H] ⁺ .

147.2 mg (0.23 mmol) of 2-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}[3-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)propyl]amino)-2-oxoethyl acetate were initially charged in 4.0 ml of ethanol, and 356.2 mg (4.59 mmol) of methylamine (40% in water) were added. The reaction mixture was stirred at 50°C overnight. The solvent was evaporated under vacuum, and the residue was co-distilled three times with toluene. The residue was purified using silica gel (eluent: dichloromethane/methanol 10:1). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 67.4 mg (63%) of the title compound.

LC-MS (Method 1): R _t = 0.91 min; MS (ESIpos): m/z = 470 [M+H] ⁺ .

Example 99

Trifluoroacetic acid/(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-N-methylbutanamide (1:1)

First, intermediate C52 was reductively alkylated with benzyl (2S)-2-{[(benzyloxy)carbonyl]amino}-4-oxobutanoate in analogy to C2. The secondary amino group was then acylated with 2-chloro-2-oxoethyl acetate as described for intermediate C27.

190 mg (0.244 mmol) of this intermediate was dissolved in 7.5 ml of ethanol and 0.35 ml of a 40% aqueous methylamine solution was added. The preparation was stirred at 50°C for 3 hours, after which the same amount of methylamine was added. After stirring at 50°C for a further 5 hours, the preparation was concentrated and the residue was purified by preparative HPLC. This yielded 78 mg (48% of theory) of the title compound.

LC-MS (Method 1): R _t = 1.32 min; MS (EIpos): m/z = 661 [M+H] ⁺ .

78 mg (0.118 mmol) of this intermediate were dissolved in 8 ml of ethanol and, after adding 15 mg of 10% palladium-activated carbon, hydrogenated at room temperature under standard hydrogen pressure for 3 minutes. The catalyst was then filtered off, the solvent removed under vacuum, and the product purified by preparative HPLC. After lyophilization from acetonitrile/water, 33 mg (44% of theory) of the title compound were obtained.

LC-MS (Method 1): R _t = 0.88 min; MS (ESIpos): m/z = 527 (M+H) ⁺ .

¹ H NMR (500 MHz, DMSO-d ₆ ): d = 8.1 (m, 1H), 8.0 (m, 3H), 7.9 (m, 1H),7.65 (m, 1H), 7.5 (s, 1H), 7.15-7.35 (m, 5H) 7.0 (m, 1H), 6.85 (m, 1H), 5.6 (s, 1H), 4.9 and 5.2 (2d, 2H), 4.02 and 4.22 (2d, 2H), 3.2-3.5 (m, 6H), 0.7 and 1.46 (2m, 2H), 0.8 (s, 9H).

Example 100

Trifluoroacetic acid/N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-N5-carbamoyl-L-ornithinamide (1:1)

100.0 mg (0.21 mol) of N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide (Intermediate C40) and 109.8 mg (0.28 mmol) of N5-carbamoyl-N2-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-ornithine were initially charged in 5.0 ml of acetonitrile, and 137.3 mg (1.06 mmol) of N , N -diisopropylethylamine and 175.8 mg (0.28 mmol) of T3P were added. The reaction mixture was stirred at room temperature overnight. The reaction mixture was partitioned between saturated ammonium chloride solution and ethyl acetate. The organic phase was washed twice with water and once with saturated NaCl solution. After drying over magnesium sulfate, the solvent was evaporated under vacuum. The residue was purified by preparative RP-HPLC (column: Reprosil 250x30; 10µ, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 118.9 mg (66%) of the compound N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-N5-carbamoyl-N2-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-ornithinamide.

LC-MS (Method 1): R _t = 1.31 min; MS (ESIpos): m/z = 850 [M+H] ⁺ .

Initially, 97.3 mg (0.11 mmol) of N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-N5-carbamoyl-N2-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-ornithinamide was charged in 4.0 ml of DMF, and 194.9 mg (2.29 mmol) of piperidine was added. The reaction mixture was stirred at room temperature for 2 hours and then neutralized with HOAc. The reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 250x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 101.4 mg of the title compound.

LC-MS (Method 1): R _t = 0.87 min; MS (ESIpos): m/z = 628 [M+H] ⁺ .

Example 101

Trifluoroacetic acid/L-valyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-N5-carbamoyl-L-ornithinamide (1:1)

96.2 mg (0.13 mmol) of trifluoroacetic acid/N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-N5-carbamoyl-L-ornithinamide (1:1) (Example 100) and 40.8 mg (0.13 mmol) of N-(tert-butoxycarbonyl)-L-valine 2,5-dioxopyrrolidin-1-yl ester were initially charged in 2.0 ml of DMF, and 39.4 mg (0.39 mmol) of 4-methylmorpholine were added. The reaction mixture was stirred at room temperature overnight. An additional 59.1 mg (0.59 mmol) of 4-methylmorpholine was added and stirred at room temperature overnight. The reaction mixture was partitioned between ethyl acetate and saturated ammonium chloride solution. The organic phase was washed once with saturated NaCl solution and dried over magnesium sulfate. The solvent was evaporated under vacuum, and the residue was purified by preparative RP-HPLC (column: Reprosil 250x30; 10µm, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 74.1 mg (69%) of the compound N-(tert-butoxycarbonyl)-L-valyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(glycolyl)amino]propyl}-N5-carbamoyl-L-ornithinamide.

LC-MS (Method 1): R _t = 1.27 min; MS (ESIpos): m/z = 827 [M+H] ⁺ .

68.1 mg (0.08 mmol) of N-(tert-butoxycarbonyl)-L-valyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-N5-carbamoyl-L-ornithinamide was dissolved in 4.0 ml of dichloromethane and 187.8 mg (1.65 mmol) of TFA was added. The reaction mixture was stirred at room temperature overnight, and an additional 187.8 mg (1.65 mmol) of TFA was added, followed by stirring at room temperature again overnight. The solvent was evaporated under vacuum, and the residue was purified by preparative RP-HPLC (column: Reprosil 250x30; 10µm, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 67.2 mg (97%) of the title compound.

LC-MS (Method 1): R _t = 0.97 min; MS (ESIpos): m/z = 727 [M+H] ⁺ .

Example 102

N-(3-Aminopropyl)-N-{(1S)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide

The synthesis was carried out similarly to the synthesis of Compound Example 98 using the corresponding S-isomer intermediate.

LC-MS (Method 1): R _t = 0.92 min; MS (ESIpos): m/z = 470 [M+H] ⁺ .

Example 103A

Here, 5.0 mg of cetuximab (c = 15.33 mg/ml) in PBS was used for coupling to intermediate F103, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 0.9 mg/ml

Drug/mAb ratio: 2.2.

Example 103B

Here, 5 mg of anti-TWEAKR AK-1 (c = 12.9 mg/ml) in PBS was used to couple with intermediate F103, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 0.95 mg/ml

Drug/mAb ratio: 2.0.

Example 103E

Here, 5.0 mg of trastuzumab antibody (c = 8.23 mg/ml) in PBS was used for coupling to intermediate F103, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.05 mg/ml

Drug/mAb ratio: 2.7.

Example 104A

Here, 5 mg of cetuximab (c = 15.33 mg/ml) in PBS was used for coupling to intermediate F104, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.95 mg/ml

Drug/mAb ratio: 3.7.

Example 104B

Here, 35 mg of anti-TWEAKR AK-1 (c = 12.9 mg/ml) in PBS was used to couple with intermediate F104, and the preparation was concentrated by ultracentrifugation after dextran gel purification, rediluted with PBS, and reconcentrated. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 10.93 mg/ml

Drug/mAb ratio: 3.2.

Example 104E

Here, 5.0 mg of trastuzumab antibody (c = 8.23 mg/ml) in PBS was used for coupling to intermediate F104, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.11 mg/ml

Drug/mAb ratio: 2.8.

Example 104I

Here, 5.0 mg of nimotuzumab (c = 13.1 mg/ml) in PBS was used for conjugation with intermediate F104, and the formulation was concentrated by ultracentrifugation after purification on a dextran gel and rediluted. Some of the ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.89 mg/ml

Drug/mAb ratio: 3.5.

Example 104H

Here, 5.0 mg of panitumumab (c = 12 mg/ml) in PBS was coupled to intermediate F104. The reduction time with TCEP was increased to 4 hours, and the stirring time for ADC coupling was increased to 20 hours. The reaction was then concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also be present as hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.79 mg/ml

Drug/mAb ratio: 2.2.

Example 105A

Here, 5 mg of cetuximab (c = 5.9 mg/ml) in PBS was used for coupling to intermediate F105, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.83 mg/ml

Drug/mAb ratio: 2.7.

Example 105B

Here, 5 mg of anti-TWEAKR AK-1 (c = 12.23 mg/ml) in PBS was used to couple with intermediate F105, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.01 mg/ml

Drug/mAb ratio: 2.6.

Example 106A

Here, 5 mg of cetuximab (c = 15.33 mg/ml) in PBS was used for conjugation with intermediate F106, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.91 mg/ml

Drug/mAb ratio: 3.3.

Example 106B

Here, 5 mg of anti-TWEAKR AK-1 (c = 12.87 mg/ml) in PBS was used to couple with intermediate F106, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.76 mg/ml

Drug/mAb ratio: 3.0.

Example 106E

Here, 5 mg of trastuzumab (c = 8.23 mg/ml) in PBS was used to couple with intermediate F106, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.5 mg/ml

Drug/mAb ratio: 2.4.

Example 107A

Here, 5 mg of cetuximab (c = 12.3 mg/ml) in PBS was used for coupling to intermediate F107, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.16 mg/ml

Drug/mAb ratio: 3.3.

Example 107B

Here, 5 mg of anti-TWEAKR AK-1 (c = 12.9 mg/ml) in PBS was used to couple with intermediate F107, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.9 mg/ml

Drug/mAb ratio: 3.1.

Example 107E

Here, 5 mg of trastuzumab (c = 8.23 mg/ml) in PBS was used for coupling to intermediate F107, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.48 mg/ml

Drug/mAb ratio: 2.9.

Example 108A

Here, 5 mg of cetuximab (c = 15.3 mg/ml) in PBS was used for conjugation with intermediate F108, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.98 mg/ml

Drug/mAb ratio: 3.2.

Example 108B

Here, 5 mg of anti-TWEAKR AK-1 (c = 12.9 mg/ml) in PBS was used to couple with intermediate F108, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.22 mg/ml

Drug/mAb ratio: 2.1.

Example 109A

Here, 5 mg of cetuximab (c = 12.33 mg/ml) in PBS was conjugated to intermediate F109. After TCEP reduction, conjugation with the antibody was performed with stirring overnight, followed by further workup via Sephadex purification. Following Sephadex purification, the reaction was concentrated by ultracentrifugation and rediluted with PBS.

Protein concentration: 2.1 mg/ml

Drug/mAb ratio: 3.1.

Example 109B

Here, 5 mg of anti-TWEAKR AK-1 (c = 34.4 mg/ml) in PBS was used for coupling with intermediate F109. After TCEP reduction, coupling with the antibody was performed with stirring overnight, followed by further workup via Sephadex purification. Following Sephadex purification, the reaction was concentrated by ultracentrifugation and rediluted with PBS.

Protein concentration: 1.63 mg/ml

Drug/mAb ratio: 2.7.

Example 110A

Here, 5 mg of cetuximab (c = 15.33 mg/ml) in PBS was coupled to intermediate F110. After TCEP reduction, the antibody was coupled with stirring overnight and then further worked up by Sephadex purification. Following Sephadex purification, the reaction was concentrated by ultracentrifugation and rediluted with PBS.

Protein concentration: 1.86 mg/ml

Drug/mAb ratio: 3.8.

Example 110B

Here, 5 mg of anti-TWEAKR AK-1 (c = 12.9 mg/ml) in PBS was used for coupling with intermediate F110. After TCEP reduction, coupling with the antibody was performed with stirring overnight, followed by further workup via Sephadex purification. Following Sephadex purification, the reaction was concentrated by ultracentrifugation and rediluted with PBS.

Protein concentration: 1.64 mg/ml

Drug/mAb ratio: 3.7.

Example 110E

Here, 5 mg of trastuzumab (c = 8.23 mg/ml) in PBS was coupled to intermediate F110. After TCEP reduction, the antibody was coupled with stirring overnight and then further worked up by Sephadex purification. Following Sephadex purification, the reaction was concentrated by ultracentrifugation and rediluted with PBS.

Protein concentration: 2.23 mg/ml

Drug/mAb ratio: 2.4.

Example 111A

Here, 5 mg of cetuximab (c = 12.33 mg/ml) in PBS was used for conjugation with intermediate F111, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.4 mg/ml

Drug/mAb ratio: 3.

Example 111B

Here, 5 mg of anti-TWEAKR AK-1 (c = 34.4 mg/ml) in PBS was used to couple with intermediate F111, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.71 mg/ml

Drug/mAb ratio: 2.5.

Example 112A

Here, 5 mg of cetuximab (c = 12.33 mg/ml) in PBS was used for conjugation with intermediate F122, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.2 mg/ml

Drug/mAb ratio: 3.

Example 112B

Here, 5 mg of anti-TWEAKR AK-1 (c = 34.4 mg/ml) in PBS was used to couple with intermediate F112, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.39 mg/ml

Drug/mAb ratio: 2.1.

Example 113A

Here, 5 mg of cetuximab (c = 12.33 mg/ml) in PBS was used for coupling to intermediate F113, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.95 mg/ml

Drug/mAb ratio: 2.2.

Example 113B

Here, 5 mg of anti-TWEAKR AK-1 (c = 34.4 mg/ml) in PBS was used to couple with intermediate F113, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.8 mg/ml

Drug/mAb ratio: 2.2.

Example 114A

Here, 5 mg of cetuximab (c = 12.33 mg/ml) in PBS was used for conjugation with intermediate F114, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.07 mg/ml

Drug/mAb ratio: 2.8.

Example 114B

Here, 5 mg of anti-TWEAKR AK-1 (c = 34.4 mg/ml) in PBS was used to couple with intermediate F114, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.9 mg/ml

Drug/mAb ratio: 2.4.

Example 115A

Here, 5 mg of cetuximab (c = 12.33 mg/ml) in PBS was used for conjugation with intermediate F115, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.18 mg/ml

Drug/mAb ratio: 3.7.

Example 115B

Here, 5 mg of anti-TWEAKR AK-1 (c = 34.4 mg/ml) in PBS was used to couple with intermediate F115, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.0 mg/ml

Drug/mAb ratio: 3.0.

Example 116A

Here, 5 mg of cetuximab (c = 12.33 mg/ml) in PBS was used for conjugation with intermediate F116, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.03 mg/ml

Drug/mAb ratio: 4.4.

Example 116B

Here, 5 mg of anti-TWEAKR AK-1 (c = 34.4 mg/ml) in PBS was used to couple with intermediate F116, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.96 mg/ml

Drug/mAb ratio: 2.9.

Example 117A

Here, 5 mg of cetuximab (c = 12.33 mg/ml) in PBS was used for coupling to intermediate F117, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 2.02 mg/ml

Drug/mAb ratio: 2.7.

Example 117B

Here, 5 mg of anti-TWEAKR AK-1 (c=34.4 mg/ml) in PBS were used for coupling to intermediate F117, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.77 mg/ml

Drug/mAb ratio: 2.7.

Example 118A

Here, 5 mg of cetuximab (c = 26.84 mg/ml) in PBS was used for coupling to intermediate F118, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 2.38 mg/ml

Drug/mAb ratio: 3.6.

Example 118B

Here, 5 mg of anti-TWEAKR AK-1 (c = 12.87 mg/ml) in PBS were used for coupling to intermediate F118, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.14 mg/ml

Drug/mAb ratio: 2.9.

Example 118E

Here, 5 mg of trastuzumab (c = 8.23 mg/ml) in PBS was used for coupling to intermediate F118, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 2.27 mg/ml

Drug/mAb ratio: 3.

Example 119A

Here, 5 mg of cetuximab (c = 26.8 mg/ml) in PBS was conjugated to intermediate F119. After TCEP reduction, conjugation with the antibody was performed with stirring overnight, followed by further workup via Sephadex purification. Following Sephadex purification, the reaction was concentrated by ultracentrifugation and rediluted with PBS.

Protein concentration: 2.14 mg/ml

Drug/mAb ratio: 3.9.

Example 119B

Here, 5 mg of anti-TWEAKR AK-1 (c = 12.87 mg/ml) in PBS was conjugated to intermediate F119. After TCEP reduction, conjugation with the antibody was performed with stirring overnight, followed by further workup via Sephadex purification. Following Sephadex purification, the preparation was concentrated by ultracentrifugation and rediluted with PBS.

Protein concentration: 0.91 mg/ml

Drug/mAb ratio: 4.1.

Example 119E

Here, 5 mg of trastuzumab (c = 13.5 mg/ml) in PBS was conjugated to intermediate F119. After TCEP reduction, conjugation with the antibody was performed with stirring overnight, followed by further workup via Sephadex purification. Following Sephadex purification, the preparation was concentrated by ultracentrifugation and rediluted with PBS.

Protein concentration: 1.69 mg/ml

Drug/mAb ratio: 4.4.

Example 120A

Here, 5 mg of cetuximab (c = 26.84 mg/ml) in PBS was used for coupling to intermediate F120, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.95 mg/ml

Drug/mAb ratio: 3.4.

Example 120B

Here, 5 mg of anti-TWEAKR AK-1 (c = 12.87 mg/ml) in PBS was used for coupling to intermediate F120, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.71 mg/ml

Drug/mAb ratio: 3.3.

Example 121A

Here, 5 mg of cetuximab (c = 26.84 mg/ml) in PBS was used for conjugation with intermediate F121, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.1 mg/ml

Drug/mAb ratio: 3.2.

Example 121B

Here, 5 mg of anti-TWEAKR AK-1 (c = 12.87 mg/ml) in PBS was used for coupling to intermediate F121, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.88 mg/ml

Drug/mAb ratio: 3.4.

Example 122A

Here, 5 mg of cetuximab (c = 26.84 mg/ml) in PBS was used for coupling to intermediate F122, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.78 mg/ml

Drug/mAb ratio: 3.2.

Example 122B

Here, 5 mg of anti-TWEAKR AK-1 (c = 12.87 mg/ml) in PBS was used to couple with intermediate F122, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.64 mg/ml

Drug/mAb ratio: 3.4.

Example 123A

Here, 5 mg of cetuximab (c = 26.84 mg/ml) in PBS was used for conjugation with intermediate F123, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.98 mg/ml

Drug/mAb ratio: 2.9.

Example 123B

Here, 5 mg of anti-TWEAKR AK-1 (c = 12.87 mg/ml) in PBS was used to couple with intermediate F123, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.84 mg/ml

Drug/mAb ratio: 3.0.

Example 124A

Here, 5 mg of cetuximab (c = 26.84 mg/ml) in PBS was used for conjugation with intermediate F124, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.93 mg/ml

Drug/mAb ratio: 2.8.

Example 124B

Here, 5 mg of anti-TWEAKR AK-1 (c = 12.87 mg/ml) in PBS was used for coupling with intermediate F124, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.84 mg/ml

Drug/mAb ratio: 3.0.

Example 125A

Here, 5 mg of cetuximab (c = 26.84 mg/ml) in PBS was used for conjugation with intermediate F125, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.14 mg/ml

Drug/mAb ratio: 2.9.

Example 125B

Here, 5 mg of anti-TWEAKR AK-1 (c = 12.87 mg/ml) in PBS was used to couple with intermediate F125, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.83 mg/ml

Drug/mAb ratio: 2.3.

Example 126A

Here, 5 mg of cetuximab (c = 26.84 mg/ml) in PBS was conjugated to intermediate F126. After TCEP reduction, conjugation with the antibody was performed with stirring overnight, followed by further workup via Sephadex purification. Following Sephadex purification, the reaction was concentrated by ultracentrifugation and rediluted with PBS.

Protein concentration: 1.89 mg/ml

Drug/mAb ratio: 2.5.

Example 126B

Here, 5 mg of anti-TWEAKR AK-1 (c = 12.9 mg/ml) in PBS was used for coupling with intermediate F126. After TCEP reduction, coupling with the antibody was performed with stirring overnight, followed by further workup via Sephadex purification. Following Sephadex purification, the reaction was concentrated by ultracentrifugation and rediluted with PBS.

Protein concentration: 1.62 mg/ml

Drug/mAb ratio: 2.8.

Example 126E

Here, 5 mg of trastuzumab (c = 8.23 mg/ml) in PBS was conjugated to intermediate F126. After TCEP reduction, conjugation with the antibody was performed with stirring overnight, followed by further workup via Sephadex purification. Following Sephadex purification, the reaction was concentrated by ultracentrifugation and rediluted with PBS.

Protein concentration: 1.93 mg/ml

Drug/mAb ratio: 1.9.

Example 127A

Here, 5 mg of cetuximab (c = 26.84 mg/ml) in PBS was used for conjugation with intermediate F127, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.54 mg/ml

Drug/mAb ratio: 3.3.

Example 127B

Here, 5 mg of anti-TWEAKR AK-1 (c = 12.9 mg/ml) in PBS was used to couple with intermediate F127, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.62 mg/ml

Drug/mAb ratio: 3.3.

Example 127E

Here, 5.0 mg of trastuzumab antibody (c = 13.5 mg/ml) in PBS was used for coupling to intermediate F127, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.07 mg/ml

Drug/mAb ratio: 3.6.

Example 128A

Here, 5 mg of cetuximab (c = 26.84 mg/ml) in PBS was used for coupling to intermediate F128, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.99 mg/ml

Drug/mAb ratio: 2.7.

Example 128B

Here, 5 mg of anti-TWEAKR AK-1 (c=12.9 mg/ml) in PBS were used for coupling to intermediate F128, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.99 mg/ml

Drug/mAb ratio: 3.4.

Example 129A

Here, 5 mg of cetuximab (c = 26.84 mg/ml) in PBS was used for coupling to intermediate F129, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 2.28 mg/ml

Drug/mAb ratio: 2.9.

Example 129B

Here, 5 mg of anti-TWEAKR AK-1 (c = 12.9 mg/ml) in PBS were used for coupling to intermediate F129, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 2.06 mg/ml

Drug/mAb ratio: 3.2.

Example 129E

Here, 5 mg of trastuzumab (c = 13.5 mg/ml) in PBS was used for coupling to intermediate F129, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 2.0 mg/ml

Drug/mAb ratio: 3.4.

Example 130

S-(1-{2-[(N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl)amino]ethyl}-2,5-dioxopyrrolidin-3-yl)-L-cysteine/trifluoroacetic acid (1:1)

4.2 mg (5 μmol) of intermediate F32 were dissolved in 2 ml of DCM/water 10:1 and 1.8 mg (15 μmol) of L-cysteine were added. The reaction mixture was stirred at room temperature for 2 hours, then concentrated under vacuum and purified by preparative HPLC.

LC-MS (Method 1): R _t = 0.8 min; MS (EIpos): m/z = 829 [M+H] ⁺ .

Example 131

S-[1-(2-{[2-({(2S)-2-amino-4-[{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]amino}-2-oxoethyl)-2,5-dioxopyrrolidin-3-yl]-L-cysteine/trifluoroacetic acid (1:1)

5 mg (6 μmol) of intermediate F87 were placed in 1 mL of DMF and 7.5 mg (62 μmol) of L-cysteine were added. The reaction mixture was stirred at room temperature for 20 h, then concentrated under vacuum and purified by preparative HPLC.

LC-MS (Method 1): R _t = 0.81 min; MS (EIpos): m/z = 815 [M+H] ⁺ .

Example 132

N-{(2S)-2-amino-4-[{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl-L-alanyl-N-[4-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)phenyl]-N ⁵ -carbamoyl-L-ornithinamide/trifluoroacetic acid (1:1)

5 mg (5 μmol) of Intermediate F89 was dissolved in 2 mL of DMF/water (10:1) and 1.7 mg (14 μmol) of L-cysteine was added. The reaction mixture was stirred at room temperature for 1 hour and then concentrated under vacuum. The residue was taken up in acetonitrile/water (1:1) and adjusted to pH 2 using TFA, then concentrated under vacuum and purified by preparative HPLC. Concentration of the appropriate fractions and lyophilization of the residue from acetonitrile/water yielded 3 mg of the title compound as a white foam.

LC-MS (Method 4): R _t = 0.93 min; MS (EIpos): m/z = 1105 [M+H] ⁺ .

Example 133

S-(1-{2-[(N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl)amino]ethyl}-2,5-dioxopyrrolidin-3-yl)-L-cysteine/trifluoroacetic acid (1:1)

1.6 mg (2 μmol) of intermediate F84 was placed in 1.5 ml of DMF/water (10:1) and 0.74 mg (6 μmol) of L-cysteine was added. The reaction mixture was stirred at room temperature for 10 minutes and then concentrated under vacuum. The residue was taken up in acetonitrile/water (1:1) and adjusted to pH 2 using TFA, then concentrated under vacuum and purified by preparative HPLC. Concentration of the appropriate fractions and lyophilization of the residue from acetonitrile/water yielded 1.9 mg (89% of theory) of the title compound as a white foam.

LC-MS (Method 1): R _t = 0.8 min; MS (EIpos): m/z = 828 [M+H] ⁺ .

Example 134

N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexanoyl]-L-valyl-N ⁵ -carbamoyl-L-ornithine-N ⁶ -{(2S)-2-amino-4-[{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butyryl}-L-lysine/trifluoroacetic acid (1:1)

3.8 mg (3 μmol) of intermediate F90 were placed in 1.5 ml of DMF/water (10:1) and 1.2 mg (9 μmol) of L-cysteine was added. The reaction mixture was stirred at room temperature for 15 minutes and then concentrated under vacuum. The residue was taken up in acetonitrile/water (1:1), reconcentrated, and then purified by preparative HPLC. Concentration of the appropriate fractions and lyophilization of the residue from acetonitrile/water yielded 2.3 mg (56% of theory) of the title compound as a white foam.

LC-MS (Method 4): R _t = 1.0 min; MS (EIpos): m/z = 1213 [M+H] ⁺ .

Example 135

S-[1-(2-{[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]amino}-2-oxoethyl)-2,5-dioxopyrrolidin-3-yl]-L-cysteine/trifluoroacetic acid (1:1)

1.8 mg (2 μmol) of intermediate F104 were placed in 1 ml of DMF and 2.7 mg (22 μmol) of L-cysteine were added. The reaction mixture was stirred at room temperature for 20 hours, then concentrated under vacuum and purified by preparative HPLC. This left 0.6 mg (26% of theory) of the title compound as a colorless foam.

LC-MS (Method 1): R _t = 0.80 min; MS (EIpos): m/z = 814 [M+H] ⁺ .

Example 136

S-(2-{[2-({(2S)-2-amino-4-[{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]amino}-2-oxoethyl)-L-cysteine/trifluoroacetic acid (1:1)

3.3 mg (4 μmol) of intermediate F109 were placed in 2 ml of DMF/water (10:1) and 1.5 mg (13 μmol) of L-cysteine was added. The reaction mixture was stirred at room temperature for 30 minutes and then concentrated under vacuum. The residue was taken up in acetonitrile/water (1:1), reconcentrated, and then purified by preparative HPLC. Concentration of the appropriate fractions and lyophilization of the residue from acetonitrile/water yielded 1.9 mg (55% of theory) of the title compound as a white foam.

LC-MS (Method 1): R _t = 0.75 min; MS (EIpos): m/z = 718 [M+H] ⁺ .

Example 137

S-{1-[6-(2-{(2S)-2-amino-4-[{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}hydrazino)-6-oxohexyl]-2,5-dioxopyrrolidin-3-yl}-L-cysteine/trifluoroacetic acid (1:1)

3.2 mg (4 μmol) of intermediate F117 were placed in 2 ml of DMF/water (10:1) and 1.6 mg (13 μmol) of L-cysteine were added. The reaction mixture was stirred at room temperature for 30 minutes and then concentrated under vacuum. The residue was taken up in acetonitrile/water (1:1), reconcentrated, and then purified by preparative HPLC. Concentration of the appropriate fractions and lyophilization of the residue from acetonitrile/water yielded 2 mg (47% of theory) of the title compound as a white foam.

LC-MS (Method 1): R _t = 0.76 min; MS (EIpos): m/z = 843 [M+H] ⁺ .

Example 138

N-[19-(3(R/S)-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecanoyl]-R/S-{2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}homocysteine/trifluoroacetic acid (1:1)

6.0 mg (0.01 mmol) of R/S-{2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}-N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecanoyl]-homocysteine/trifluoroacetic acid (1:1) (Intermediate F146) was initially charged in 2.2 ml of DMF/water (10:1), 2.0 mg (0.02 mmol) of L-cysteine was added and stirred at room temperature for 10 minutes. The reaction mixture was purified directly by preparative RP-HPLC (column: Reprosil 250x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 5.5 mg (76% of theory) of the title compound.

LC-MS (Method 4): R _t = 1.07 min; MS (ESIpos): m/z = 1106 (M+H) ⁺ .

Example 139

S-{(3R/S)-1-[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]-2,5-dioxopyrrolidin-3-yl}-L-cysteine/trifluoroacetic acid (1:2)

The synthesis was carried out analogously to the synthesis of compound Example 138.

6.0 mg (0.01 mmol) trifluoroacetic acid/(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]butanamide (1:1).

2.6 mg (0.02 mmol) L-cysteine.

This gave 3.4 mg (43% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.82 min; MS (ESIpos): m/z = 757 (M+H) ⁺ .

Example 140

N-[19-(3(R/S)-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecanoyl]-R/S-{2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}homocysteine/trifluoroacetic acid (1:2)

The synthesis was carried out analogously to the synthesis of compound Example 138.

6.0 mg (0.01 mmol) trifluoroacetic acid/R/S-{2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}-N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecan-1-oyl]homocysteine (1:1) (Intermediate F149).

2.0 mg (0.02 mmol) L-cysteine.

This gave 6.1 mg (84% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.85 min; MS (ESIpos): m/z = 1107 (M+H) ⁺ .

Example 141

S-[(3R/S)-1-(2-{[6-({2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}sulfanyl)hexanoyl]amino}ethyl)-2,5-dioxopyrrolidin-3-yl]-L-cysteine/trifluoroacetic acid (1:2)

The synthesis was carried out analogously to the synthesis of compound Example 138.

10.07 mg (0.01 mmol) trifluoroacetic acid/6-({2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}sulfanyl)-N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]hexanamide (1:1) (Intermediate F143).

9.3 mg (0.08 mmol) L-cysteine.

This gave 9.2 mg (67% of theory) of the title compound.

LC-MS (Method 4): R _t = 1.05 min; MS (ESIpos): m/z = 843 (M+H) ⁺ .

Example 142A

Here, 5.0 mg of cetuximab (c = 12.33 mg/ml) in PBS was used for conjugation with intermediate F142, and the formulation was concentrated by ultracentrifugation after purification on a dextran gel and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.08 mg/ml

Drug/mAb ratio: 2.1.

Example 142B

Here, 5.0 mg of anti-TWEAKR AK-1 antibody (c = 34.42 mg/ml) in PBS was conjugated to intermediate F142, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.84 mg/ml

Drug/mAb ratio: 2.0.

Example 142E

Here, 5.0 mg of trastuzumab antibody (c = 13.50 mg/ml) in PBS was conjugated to intermediate F142, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.92 mg/ml

Drug/mAb ratio: 2.3.

Example 142I

Here, 5.0 mg of nimotuzumab (c = 13.8 mg/ml) in PBS was used for conjugation with intermediate F142, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.92 mg/ml

Drug/mAb ratio: 2.5.

Example 142H

Here, 5.0 mg of panitumumab (c = 2.1 mg/ml) in PBS was coupled to intermediate F142. Reduction with TCEP was performed for 1 hour, and the stirring time for ADC coupling was 1.5 hours. Following purification on a dextran gel, the formulation was concentrated by ultracentrifugation and rediluted with PBS. Some ADC may also be present as hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.7 mg/ml

Drug/mAb ratio: 2.2.

Example 143A

Here, 5.0 mg of cetuximab (c = 12.33 mg/ml) in PBS was conjugated to intermediate F143, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.84 mg/ml

Drug/mAb ratio: 2.5.

Example 143B

Here, 5.0 mg of anti-TWEAKR AK-1 antibody (c = 34.42 mg/ml) in PBS was conjugated to intermediate F143, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.05 mg/ml

Drug/mAb ratio: 1.6.

Example 143E

Here, 5.0 mg of trastuzumab antibody (c = 13.50 mg/ml) in PBS was conjugated to intermediate F143, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.95 mg/ml

Drug/mAb ratio: 2.3.

Example 144B

Here, 5.0 mg of anti-TWEAKR AK-1 antibody (c=12.87 mg/ml) in PBS was used for coupling to intermediate F144, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted.

Protein concentration: 1.53 mg/ml

Drug/mAb ratio: 2.0.

Example 145A

Here, 5.0 mg of cetuximab (c = 5.90 mg/ml) in PBS was conjugated to intermediate F145, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.00 mg/ml

Drug/mAb ratio: 2.0.

Example 145B

Here, 5.0 mg of anti-TWEAKR AK-1 antibody (c = 12.87 mg/ml) in PBS was conjugated to intermediate F145, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.54 mg/ml

Drug/mAb ratio: 1.9.

Example 145E

Here, 5.0 mg of trastuzumab antibody (c = 13.50 mg/ml) in PBS was conjugated to intermediate F145, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.86 mg/ml

Drug/mAb ratio: 2.4.

Example 146A

Here, 5.0 mg of cetuximab (c = 12.33 mg/ml) in PBS was used for conjugation with intermediate F146, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.02 mg/ml

Drug/mAb ratio: 2.4.

Example 146B

Here, 5.0 mg of anti-TWEAKR AK-1 antibody (c = 34.42 mg/ml) in PBS was conjugated to intermediate F146, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.87 mg/ml

Drug/mAb ratio: 2.4.

Example 146E

Here, 5.0 mg of trastuzumab antibody (c = 13.50 mg/ml) in PBS was conjugated to intermediate F146, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.92 mg/ml

Drug/mAb ratio: 2.5.

Example 147A

Here, 5.0 mg of cetuximab (c = 5.90 mg/ml) in PBS was conjugated to intermediate F147, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.04 mg/ml

Drug/mAb ratio: 2.2.

Example 147B

Here, 5.0 mg of anti-TWEAKR AK-1 antibody (c = 12.87 mg/ml) in PBS was conjugated to intermediate F147, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.31 mg/ml

Drug/mAb ratio: 1.8.

Example 147E

Here, 5.0 mg of trastuzumab antibody (c = 13.50 mg/ml) in PBS was conjugated to intermediate F147, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.86 mg/ml

Drug/mAb ratio: 2.6.

Example 148A

Here, 5.0 mg of cetuximab (c = 5.90 mg/ml) in PBS was used for conjugation with intermediate F148, and the formulation was concentrated by ultracentrifugation after purification on a dextran gel and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.11 mg/ml

Drug/mAb ratio: 2.3.

Example 148B

Here, 5.0 mg of anti-TWEAKR AK-1 antibody (c = 12.87 mg/ml) in PBS was conjugated to intermediate F148, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.95 mg/ml

Drug/mAb ratio: 2.0.

Example 148E

Here, 5.0 mg of trastuzumab antibody (c = 13.50 mg/ml) in PBS was conjugated to intermediate F148, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.62 mg/ml

Drug/mAb ratio: 2.5.

Example 149A

Here, 5.0 mg of cetuximab (c = 5.90 mg/ml) in PBS was used for conjugation with intermediate F149, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.09 mg/ml

Drug/mAb ratio: 2.3.

Example 149B

Here, 40 mg of anti-TWEAKR AK-1 (c = 34.42 mg/ml) in PBS was used to couple with intermediate F149, and the preparation was concentrated by ultracentrifugation after Sephadex purification, rediluted with PBS, and reconcentrated. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 9.61 mg/ml

Drug/mAb ratio: 2.6.

Example 149E

Here, 5.0 mg of trastuzumab antibody (c = 13.50 mg/ml) in PBS was conjugated to intermediate F149, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.93 mg/ml

Drug/mAb ratio: 2.7.

Example 150A

Here, 5.0 mg of cetuximab (c = 15.33 mg/ml) in PBS was conjugated to intermediate F150, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.91 mg/ml

Drug/mAb ratio: 2.1.

Example 150B

Here, 5.0 mg of anti-TWEAKR AK-1 antibody (c = 12.87 mg/ml) in PBS was conjugated to intermediate F150, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.81 mg/ml

Drug/mAb ratio: 2.1.

Example 150E

Here, 5.0 mg of trastuzumab antibody (c = 13.50 mg/ml) in PBS was conjugated to intermediate F150, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.84 mg/ml

Drug/mAb ratio: 2.6.

Example 151A

Here, 5.0 mg of cetuximab (c = 16.90 mg/ml) in PBS was used for conjugation with intermediate F151, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.86 mg/ml

Drug/mAb ratio: 2.3.

Example 151B

Here, 5.0 mg of anti-TWEAKR AK-1 antibody (c = 12.87 mg/ml) in PBS was conjugated to intermediate F151, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.78 mg/ml

Drug/mAb ratio: 2.2.

Example 151E

Here, 5.0 mg of trastuzumab antibody (c = 16.90 mg/ml) in PBS was conjugated to intermediate F151, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.74 mg/ml

Drug/mAb ratio: 1.9.

Example 152A

Here, 5.0 mg of cetuximab (c = 16.90 mg/ml) in PBS was conjugated to intermediate F152, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.80 mg/ml

Drug/mAb ratio: 2.3.

Example 152B

Here, 5.0 mg of anti-TWEAKR AK-1 antibody (c = 12.87 mg/ml) in PBS was conjugated to intermediate F152, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.77 mg/ml

Drug/mAb ratio: 2.3.

Example 152E

Here, 5.0 mg of trastuzumab antibody (c = 16.90 mg/ml) in PBS was conjugated to intermediate F152, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.69 mg/ml

Drug/mAb ratio: 2.9.

Example 153A

Here, 5 mg of cetuximab (c = 21.32 mg/ml) in PBS was used for coupling to intermediate F153, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.93 mg/ml

Drug/mAb ratio: 3.2.

Example 153B

Here, 5 mg of anti-TWEAKR AK-1 (c = 18.6 mg/ml) in PBS was used to couple with intermediate F153, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.71 mg/ml

Drug/mAb ratio: 2.8.

Example 154A

Here, 5 mg of cetuximab (c = 26.8 mg/ml) in PBS was used for coupling to intermediate F154, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 2.34 mg/ml

Drug/mAb ratio: 3.6.

Example 154B

Here, 5 mg of anti-TWEAKR AK-1 (c=12.9 mg/ml) in PBS were used for coupling to intermediate F154, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.83 mg/ml

Drug/mAb ratio: 3.8.

Example 155A

Here, 50 mg of cetuximab (c=8.51 mg/ml) in PBS was used for coupling to intermediate F155, and the formulation was concentrated by ultracentrifugation after Sephadex purification, rediluted with PBS and reconcentrated.

Protein concentration: 14.85 mg/ml

Drug/mAb ratio: 2.5.

Example 155B

Here, 40 mg of anti-TWEAKR AK-1 (c=18.6 mg/ml) in PBS were used for coupling to intermediate F155, and the preparation was concentrated by ultracentrifugation after Sephadex purification, rediluted with PBS and reconcentrated.

Protein concentration: 11.25 mg/ml

Drug/mAb ratio: 3.1.

Example 156A

Here, 5 mg of cetuximab (c = 21.3 mg/ml) in PBS was conjugated to intermediate F156. After TCEP reduction, conjugation with the antibody was performed with stirring overnight, followed by further workup via Sephadex purification. Following Sephadex purification, the preparation was concentrated by ultracentrifugation and rediluted with PBS.

Protein concentration: 1.83 mg/ml

Drug/mAb ratio: 3.6.

Example 156B

Here, 5 mg of anti-TWEAKR AK-1 (c=18.6 mg/ml) in PBS were used for coupling to intermediate F156, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.79 mg/ml

Drug/mAb ratio: 3.9.

Example 156E

Here, 5 mg of trastuzumab (c = 13.5 mg/ml) in PBS was used for coupling to intermediate F156, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.91 mg/ml

Drug/mAb ratio: 4.2.

Example 157

S-{1-[2-({[(1R,3S)-3-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)cyclopentyl]carbonyl}amino)ethyl]-2,5-dioxopyrrolidin-3-yl}-L-cysteine/trifluoroacetic acid (1:1)

2 mg (2 μmol) of intermediate F125 were dissolved in 2 ml of DMF/water 10:1 and 0.8 mg (6 μmol) of L-cysteine were added. The reaction mixture was stirred at room temperature for 20 h, then concentrated under vacuum and purified by preparative HPLC.

LC-MS (Method 1): R _t = 0.81 min; MS (EIpos): m/z = 868 [M+H] ⁺ .

Example 158

S-(2-{[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]amino}-2-oxoethyl)-L-cysteine/trifluoroacetic acid (1:1)

6 mg (8 μmol) of intermediate F119 was placed in 3 mL of DMF and 1.8 mg (15 μmol) of L-cysteine was added. The reaction mixture was stirred at room temperature for 6 hours and then allowed to stand at room temperature for 3 days. The reaction was then concentrated under vacuum, and the product was purified by preparative HPLC.

LC-MS (Method 1): R _t = 0.81 min; MS (ESIpos): m/z = 717 (M+H) ⁺ .

Example 159

N ⁶ -(N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(glycoyl)amino]butyryl}-D-alanyl)-L-lysine/trifluoroacetic acid (1:1)

First, N6-D-alanyl-N2-(tert-butoxycarbonyl)-L-lysine methyl ester was prepared from N-[(benzyloxy)carbonyl]-D-alanine 2,5-dioxopyrrolidin-1-yl ester and N2-(tert-butoxycarbonyl)-L-lysine methyl ester using classical methods of peptide chemistry.

To synthesize the title compound, 8.4 mg (0.022 mmol) of this intermediate was placed in 4 mL of DMF, and 10 mg (0.015 mmol) of intermediate C5, 11 mg of HATU, and 13 μL of N , N -diisopropylethylamine were added. After stirring at room temperature for 5 minutes, the preparation was purified by preparative HPLC. After concentration of the appropriate fractions and drying under high vacuum, the resulting intermediate was dissolved in 4 mL of methanol, 83 μL of 2M lithium hydroxide solution was added, and the preparation was stirred at room temperature overnight. An additional 167 μL of lithium hydroxide solution was added and stirred for an additional 4 hours. The mixture was then diluted with water and adjusted to pH 5 with 5% citric acid. After concentration, the residue was purified by preparative HPLC. Concentration of the appropriate fractions and lyophilization of the residue from acetonitrile/water yielded 3.5 mg (26% of theory) of the Boc-protected intermediate. Deprotection with 1 mL of trifluoroacetic acid in 2 mL of DCM yielded 3 mg (95% of theory) of the title compound.

HPLC (Method 11): R _t = 1.76 min;

LC-MS (Method 1): R _t = 0.75 min; MS (ESIpos): m/z = 714 (M+H) ⁺ .

Example 160

N ⁶ -(N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl)-N ² -{N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexanoyl]-L-valyl-L-alanyl}-L-lysine/trifluoroacetic acid (1:1)

4 mg (3 μmol) of intermediate F155 were dissolved in 2.5 mL of DMF/water (10:1) and 1.2 mg (10 μmol) of L-cysteine were added. The reaction mixture was stirred at room temperature for 30 minutes, then concentrated under vacuum, taken up in acetonitrile/water (1:1), and purified by preparative HPLC.

LC-MS (Method 1): R _t = 0.81 min; MS (EIpos): m/z = 1197 [M+H] ⁺ .

Example 161

N-[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]-L-glutamine/trifluoroacetic acid (1:1)

First, trifluoroacetic acid/benzyl N-(2-aminoethyl)-N ² -[(benzyloxy)carbonyl]-L-glutamate (1:1) was prepared using classical methods of peptide chemistry. This intermediate was then coupled with intermediate C58 in the presence of HATU. Subsequently, the benzyloxycarbonyl protecting group and the benzyl ester were first removed by hydrogenolytic cleavage, followed by removal of the 2-(trimethylsilyl)ethoxycarbonyl protecting group using zinc chloride.

LC-MS (Method 6): R _t = 1.91 min; MS (EIpos): m/z = 685 [M+H] ⁺ .

Example 162

N ⁶ -(N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoyl)amino]butyryl}-β-alanyl)-L-lysine/trifluoroacetic acid (1:1)

First, trifluoroacetic acid/N2-[(benzyloxy)carbonyl]-L-lysine 2-(trimethylsilyl)ethyl ester (1:1) was prepared using classical protecting group manipulations known in peptide chemistry. This intermediate was then coupled with intermediate C61 in the presence of HATU. Subsequently, the 2-(trimethylsilyl)ethoxycarbonyl protecting group and 2-(trimethylsilyl)ethyl ester were first cleaved using zinc chloride. Finally, the title compound was obtained by hydrogenolytic cleavage of the benzyloxycarbonyl protecting group and purification by preparative HPLC.

HPLC (Method 11): R _t = 1.65 min;

LC-MS (Method 1): R _t = 0.76 min; MS (EIpos): m/z = 713 [M+H] ⁺ .

Example 163A

Here, 5 mg of cetuximab (c = 11.3 mg/ml) in PBS was used for coupling to intermediate F163, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 2.02 mg/ml

Drug/mAb ratio: 3.3.

Example 163B

Here, 5 mg of anti-TWEAKR AK-1 (c=12.9 mg/ml) in PBS were used for coupling to intermediate F163, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.76 mg/ml

Drug/mAb ratio: 2.5.

Example 163H

Here, 5.0 mg of panitumumab (c = 10 mg/ml) in PBS was coupled to intermediate F163. The reduction time with TCEP was 30 minutes, and the stirring time for ADC coupling was 2 hours. After purification on Sephadex, the formulation was concentrated by ultracentrifugation and rediluted with PBS.

Protein concentration: 1.61 mg/ml

Drug/mAb ratio: 2.6.

Example 164A

Here, 5 mg of cetuximab (c = 16.9 mg/ml) in PBS was used for coupling to intermediate F164, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted.

Protein concentration: 2.15 mg/ml

Drug/mAb ratio: 3.5.

Example 164B

Here, 30 mg of anti-TWEAKR AK-1 (c=18.6 mg/ml) in PBS were used for coupling to intermediate F164, and the preparation was concentrated by ultracentrifugation after Sephadex purification, rediluted with PBS and reconcentrated.

Protein concentration: 14.8 mg/ml

Drug/mAb ratio: 2.8.

Example 164E

Here, 5 mg of trastuzumab (c = 13.5 mg/ml) in PBS was used for coupling to intermediate F164, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 2.11 mg/ml

Drug/mAb ratio: 3.8.

Example 165A

Here, 5 mg of cetuximab (c = 16.9 mg/ml) in PBS was used for coupling to intermediate F165, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.93 mg/ml

Drug/mAb ratio: 3.4.

Example 165B

Here, 40 mg of anti-TWEAKR AK-1 (c=12.9 mg/ml) in PBS were used for coupling to intermediate F165, and the preparation was concentrated by ultracentrifugation after Sephadex purification, rediluted with PBS and reconcentrated.

Protein concentration: 12.02 mg/ml

Drug/mAb ratio: 3.3.

Example 165E

Here, 5 mg of trastuzumab (c = 13.5 mg/ml) in PBS was used for coupling to intermediate F165, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.94 mg/ml

Drug/mAb ratio: 3.5.

Example 166A

Here, 5 mg of cetuximab (c = 21.3 mg/ml) in PBS was used for coupling to intermediate F166, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 2.0 mg/ml

Drug/mAb ratio: 3.0.

Example 166B

Here, 5 mg of anti-TWEAKR AK-1 (c = 18.6 mg/ml) in PBS were used for coupling to intermediate F166, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.76 mg/ml

Drug/mAb ratio: 3.4.

Example 166E

Here, 5 mg of trastuzumab (c = 13.5 mg/ml) in PBS was used for coupling to intermediate F166, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 2.01 mg/ml

Drug/mAb ratio: 3.6.

Example 167A

Here, 5 mg of cetuximab (c = 16.9 mg/ml) in PBS was used for coupling to intermediate F167, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 2.05 mg/ml

Drug/mAb ratio: 3.0.

Example 167B

Here, 5 mg of anti-TWEAKR AK-1 (c=12.9 mg/ml) in PBS were used for coupling to intermediate F167, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.76 mg/ml

Drug/mAb ratio: 2.9.

Example 167E

Here, 5 mg of trastuzumab (c = 13.5 mg/ml) in PBS was used for coupling to intermediate F167, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.9 mg/ml

Drug/mAb ratio: 3.6.

Example 168A

Here, 5 mg of cetuximab (c = 11.3 mg/ml) in PBS was used for coupling to intermediate F168, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.94 mg/ml

Drug/mAb ratio: 3.0.

Example 168B

Here, 5 mg of anti-TWEAKR AK-1 (c=12.9 mg/ml) in PBS were used for coupling to intermediate F168, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.33 mg/ml

Drug/mAb ratio: 2.8.

Example 168E

Here, 5 mg of trastuzumab (c = 13.5 mg/ml) in PBS was used for coupling to intermediate F168, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.89 mg/ml

Drug/mAb ratio: 3.0.

Example 168H

Here, 5.0 mg of panitumumab (c = 10 mg/ml) in PBS was coupled to intermediate F168. The reduction time with TCEP was 4 hours, and the stirring time for ADC coupling was 20 hours. After Sephadex purification, the reaction was concentrated by ultracentrifugation and rediluted with PBS.

Protein concentration: 1.76 mg/ml

Drug/mAb ratio: 2.8.

Example 169A

Here, 5 mg of cetuximab (c = 16.9 mg/ml) in PBS was used for coupling to intermediate F169, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.98 mg/ml

Drug/mAb ratio: 3.4.

Example 169B

Here, 40 mg of anti-TWEAKR AK-1 (c=18.6 mg/ml) in PBS were used for coupling to intermediate F169, and the preparation was concentrated by ultracentrifugation after Sephadex purification, rediluted with PBS and reconcentrated.

Protein concentration: 11.2 mg/ml

Drug/mAb ratio: 2.4.

Example 169E

Here, 5 mg of trastuzumab (c = 13.5 mg/ml) in PBS was used for coupling to intermediate F169, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.95 mg/ml

Drug/mAb ratio: 3.7.

Example 170A

Here, 5 mg of cetuximab (c = 16.9 mg/ml) in PBS was used for coupling to intermediate F170, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted.

Protein concentration: 1.91 mg/ml

Drug/mAb ratio: 2.9.

Example 170B

Here, 5 mg of anti-TWEAKR AK-1 (c=12.9 mg/ml) in PBS were used for coupling to intermediate F170, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.72 mg/ml

Drug/mAb ratio: 3.07.

Example 170E

Here, 5 mg of trastuzumab (c = 13.5 mg/ml) in PBS was used for coupling to intermediate F170, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.84 mg/ml

Drug/mAb ratio: 3.3.

Example 171A

Here, 5 mg of cetuximab (c = 11.3 mg/ml) in PBS was used for coupling to intermediate F171, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.94 mg/ml

Drug/mAb ratio: 2.5.

Example 171B

Here, 5 mg of anti-TWEAKR AK-1 (c=12.9 mg/ml) in PBS were used for coupling to intermediate F171, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.58 mg/ml

Drug/mAb ratio: 2.7.

Example 172A

Here, 5 mg of cetuximab (c = 11.3 mg/ml) in PBS was used for coupling to intermediate F172, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.96 mg/ml

Drug/mAb ratio: 3.1.

Example 172B

Here, 5 mg of anti-TWEAKR AK-1 (c=18.6 mg/ml) in PBS were used for coupling to intermediate F172, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.85 mg/ml

Drug/mAb ratio: 3.1.

Example 172E

Here, 5 mg of trastuzumab (c = 13.5 mg/ml) in PBS was used for coupling to intermediate F172, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.92 mg/ml

Drug/mAb ratio: 3.3.

Example 173A

Here, 5 mg of cetuximab (c = 11.3 mg/ml) in PBS was used for coupling to intermediate F173, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 2.1 mg/ml

Drug/mAb ratio: 3.6.

Example 173B

Here, 5 mg of anti-TWEAKR AK-1 (c=18.6 mg/ml) in PBS were used for coupling to intermediate F173, and the preparation was concentrated by ultracentrifugation after Sephadex purification, rediluted with PBS and reconcentrated.

Protein concentration: 12.26 mg/ml

Drug/mAb ratio: 3.4.

Example 173E

Here, 5 mg of trastuzumab (c = 13.5 mg/ml) in PBS was used for coupling to intermediate F173, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 2.33 mg/ml

Drug/mAb ratio: 3.9.

Example 174A

Here, 5 mg of cetuximab (c = 11.3 mg/ml) in PBS was used for coupling to intermediate F174, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 2.18 mg/ml

Drug/mAb ratio: 3.1.

Example 174B

Here, 5 mg of anti-TWEAKR AK-1 (c=18.6 mg/ml) in PBS were used for coupling to intermediate F174, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.99 mg/ml

Drug/mAb ratio: 3.1.

Example 174E

Here, 5 mg of trastuzumab (c = 13.5 mg/ml) in PBS was used for coupling to intermediate F174, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 2.03 mg/ml

Drug/mAb ratio: 3.5.

Example 175A

Here, 5 mg of cetuximab (c = 16.9 mg/ml) in PBS was used for coupling to intermediate F175, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.98 mg/ml

Drug/mAb ratio: 3.8.

Example 175B

Here, 40 mg of anti-TWEAKR AK-1 (c=18.6 mg/ml) in PBS were used for coupling to intermediate F175, and the preparation was concentrated by ultracentrifugation after Sephadex purification, rediluted with PBS and reconcentrated.

Protein concentration: 9.8 mg/ml

Drug/mAb ratio: 2.8.

Example 175E

Here, 5.0 mg of trastuzumab antibody (c = 13.5 mg/ml) in PBS was used for coupling to intermediate F175, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.97 mg/ml

Drug/mAb ratio: 4.2.

Example 176A

Here, 5 mg of cetuximab (c = 21.3 mg/ml) in PBS was used for conjugation with intermediate F176, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.98 mg/ml

Drug/mAb ratio: 2.6.

Example 176B

Here, 5 mg of anti-TWEAKR AK-1 (c = 18.6 mg/ml) in PBS was used to couple with intermediate F176, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.93 mg/ml

Drug/mAb ratio: 2.8.

Example 176E

Here, 5.0 mg of trastuzumab antibody (c = 13.5 mg/ml) in PBS was used for coupling to intermediate F176, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.85 mg/ml

Drug/mAb ratio: 3.3.

Example 177A

Here, 5 mg of cetuximab (c = 21.3 mg/ml) in PBS was used for conjugation with intermediate F177, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.96 mg/ml

Drug/mAb ratio: 2.8.

Example 177B

Here, 5 mg of anti-TWEAKR AK-1 (c = 18.6 mg/ml) in PBS was used to couple with intermediate F177, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.2 mg/ml

Drug/mAb ratio: 2.3.

Example 177E

Here, 5 mg of trastuzumab (c = 13.5 mg/ml) in PBS was used to couple with intermediate F177, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.83 mg/ml

Drug/mAb ratio: 3.1.

Example 178A

Here, 5 mg of cetuximab (c = 21.3 mg/ml) in PBS was used for coupling to intermediate F178, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.8 mg/ml

Drug/mAb ratio: 2.1.

Example 178B

Here, 5 mg of anti-TWEAKR AK-1 (c=18.6 mg/ml) in PBS were used for coupling to intermediate F178, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.45 mg/ml

Drug/mAb ratio: 2.4.

Example 178E

Here, 5 mg of trastuzumab (c = 13.5 mg/ml) in PBS was used for coupling to intermediate F178, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.8 mg/ml

Drug/mAb ratio: 2.6.

Example 179A

Here, 5 mg of cetuximab (c = 11.3 mg/ml) in PBS was used for coupling to intermediate F179, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 2.04 mg/ml

Drug/mAb ratio: 3.1.

Example 179B

Here, 5 mg of anti-TWEAKR AK-1 (c = 18.6 mg/ml) in PBS were used for coupling to intermediate F179, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.65 mg/ml

Drug/mAb ratio: 3.1.

Example 179E

Here, 5 mg of trastuzumab (c = 13.5 mg/ml) in PBS was used for coupling to intermediate F179, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.89 mg/ml

Drug/mAb ratio: 3.3.

Example 180A

Here, 5 mg of cetuximab (c = 8.51 mg/ml) in PBS was used for conjugation with intermediate F180, and the formulation was concentrated by ultracentrifugation after purification on a dextran gel and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.72 mg/ml

Drug/mAb ratio: 3.5.

Example 180B

Here, 5 mg of anti-TWEAKR AK-1 (c = 18.6 mg/ml) in PBS was used to couple with intermediate F180, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.82 mg/ml

Drug/mAb ratio: 3.4.

Example 180E

Here, 5 mg of trastuzumab (c = 13.5 mg/ml) in PBS was used for coupling to intermediate F180, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.01 mg/ml

Drug/mAb ratio: 4.7.

Example 181

N-(3-Aminopropyl)-N-{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}acetamide

Initially, 1.01 g (2.84 mmol) of (1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropan-1-amine was charged to 20 ml of 1,2-dichloroethane. 0.84 g (3.98 mmol) of sodium triacetoxyborohydride and 2.56 g (42.65 mmol) of acetic acid were added and stirred at room temperature for 5 minutes. A solution of 0.54 g (3.13 mmol) of tert-butyl (3-oxopropyl)carbamate in 5 ml of 1,2-dichloroethane was then added, and the reaction mixture was stirred overnight. The mixture was then completely evaporated, the residue taken up in ethyl acetate and filtered, and the product-containing filtrate was completely evaporated.

51.26 mg of this residue was dissolved in 0.8 mL of 1,2-dichloromethane and added to 7.85 mg (0.1 mmol) of acetyl chloride in a deep 96-well multititer plate. 25.8 mg (0.2 mmol) of N , N -diisopropylethylamine was then added and shaken overnight at room temperature. The solvent was then completely removed using a centrifugal desiccator, and 0.4 mL of 1,2-dichloroethane and 0.4 mL of trifluoroacetic acid were added to the residue, which was shaken overnight. The solvent was then completely removed using a centrifugal desiccator, and 0.8 mL of DMF was added to the residue. The filtrate was then filtered, and the target compound was isolated from the filtrate by preparative LC-MS (Method 9). Product-containing fractions were concentrated under vacuum using a centrifugal desiccator. The residues from each product fraction were dissolved in 0.6 mL of DMSO. These fractions were combined and the solvent was finally removed in a centrifugal desiccator. This yielded 12.2 mg (27% of theory; 100% purity) of the title compound.

LC-MS (Method 10): R _t = 0.95 min; MS (ESIpos): m/z = 455 [M+H] ⁺ .

The exemplary compounds shown in Table XA were prepared similarly to Example 181:

.

Example 186

N-(3-Aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-3,3,3-trifluoropropionamide

Initially, 1.0 g (2.82 mmol) of (1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropan-1-amine was charged to 20 ml of 1,2-dichloroethane. 1.4 g (3.95 mmol) of sodium triacetoxyborohydride and 2.54 g (42.32 mmol) of acetic acid were added and stirred at room temperature for 5 minutes. A solution of 0.54 g (3.10 mmol) of tert-butyl (3-oxopropyl)carbamate in 5 ml of 1,2-dichloroethane was then added, and the reaction mixture was stirred overnight. The mixture was then completely evaporated, the residue taken up in ethyl acetate and filtered, and the product-containing filtrate was completely evaporated.

51.16 mg of this residue was dissolved in 0.8 mL of 1,2-dichloromethane and added to 14.65 mg (0.1 mmol) of 3,3,3-trifluoropropionyl chloride in a deep 96-well multititer plate. 25.8 mg (0.2 mmol) of N , N -diisopropylethylamine was then added and shaken overnight at room temperature. The solvent was then completely removed using a centrifugal desiccator, and 0.4 mL of 1,2-dichloroethane and 0.4 mL of trifluoroacetic acid were added to the residue, which was shaken overnight. The solvent was then completely removed using a centrifugal desiccator, and 0.8 mL of DMF was added to the residue. The filtrate was then filtered, and the target compound was isolated from the filtrate by preparative LC-MS (Method 9). Product-containing fractions were concentrated under vacuum using a centrifugal desiccator. The residues from each product fraction were dissolved in 0.6 mL of DMSO. These fractions were combined and the solvent was finally removed in a centrifugal desiccator. This yielded 1.0 mg (2% of theory; 82% purity) of the title compound.

LC-MS (Method 10): R _t = 1.01 min MS (ESIpos): m/z = 522 [M+H] ⁺ .

The exemplary compounds shown in Table XA1 were prepared similarly to Example 186:

.

Example 192A

Here, 5 mg of cetuximab (c = 21.3 mg/ml) in PBS was used for conjugation with intermediate F192, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.968 mg/ml

Drug/mAb ratio: 2.9.

Example 192B

Here, 5 mg of anti-TWEAKR AK-1 (c = 18.6 mg/ml) in PBS was used to couple to intermediate F192, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.92 mg/ml

Drug/mAb ratio: 2.6.

Example 192E

Here, 5 mg of trastuzumab antibody (c = 13.5 mg/ml) in PBS was conjugated to intermediate F192, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.04 mg/ml

Drug/mAb ratio: 3.3.

Example 193A

Here, 5 mg of cetuximab (c = 23.1 mg/ml) in PBS was used to couple to intermediate F193, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.98 mg/ml

Drug/mAb ratio: 2.9.

Example 193B

Here, 5 mg of anti-TWEAKR AK-1 (c = 18.6 mg/ml) in PBS was used to couple to intermediate F193, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.03 mg/ml

Drug/mAb ratio: 3.2.

Example 193E

Here, 5 mg of trastuzumab antibody (c = 13.5 mg/ml) in PBS was conjugated to intermediate F193, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.61 mg/ml

Drug/mAb ratio: 3.3.

Example 194A

Here, 5 mg of cetuximab (c = 23.1 mg/ml) in PBS was used for coupling to intermediate F194, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.67 mg/ml

Drug/mAb ratio: 1.9.

Example 194B

Here, 5.0 mg of anti-TWEAKR AK-1 antibody (c=18.6 mg/ml) in PBS was used for coupling to intermediate F194, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 0.99 mg/ml

Drug/mAb ratio: 3.8.

Example 194E

Here, 5.0 mg of trastuzumab antibody (c = 13.5 mg/ml) in PBS was used for coupling to intermediate F194, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS.

Protein concentration: 1.39 mg/ml

Drug/mAb ratio: 2.4.

Example 195A

Here, 5 mg of cetuximab (c = 23.1 mg/ml) in PBS was used to couple to intermediate F195, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.79 mg/ml

Drug/mAb ratio: 3.2.

Example 195B

Here, 5 mg of anti-TWEAKR AK-1 (c = 18.6 mg/ml) in PBS was used to couple with intermediate F195, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.53 mg/ml

Drug/mAb ratio: 3.3.

Example 195E

Here, 5.0 mg of trastuzumab antibody (c = 13.5 mg/ml) in PBS was used for coupling to intermediate F195, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.91 mg/ml

Drug/mAb ratio: 3.4.

Example 196A

Here, 5 mg of cetuximab (c = 23.1 mg/ml) in PBS was used to couple to intermediate F196, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.85 mg/ml

Drug/mAb ratio: 3.0.

Example 196B

Here, 5 mg of anti-TWEAKR AK-1 (c = 18.6 mg/ml) in PBS was used to couple with intermediate F196, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.73 mg/ml

Drug/mAb ratio: 3.1.

Example 196E

Here, 5.0 mg of trastuzumab antibody (c = 13.5 mg/ml) in PBS was used for coupling to intermediate F196, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.86 mg/ml

Drug/mAb ratio: 3.4.

Example 197

(2S)-2-Amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butyric acid hydrochloride (1:1)

150 mg (0.2 mmol) of intermediate C53 was dissolved in 15 ml of DMF and 2.29 g (20.39 mmol) of DABCO was added. The preparation was treated in an ultrasonic bath for 30 minutes. 1.17 ml of acetic acid was then added to the pH of the mixture, which was then concentrated under vacuum. The residue was purified by preparative HPLC and the appropriate fractions were concentrated under vacuum at room temperature. The residue was taken up in acetonitrile/water (1:1), 5 ml of 4N hydrochloric acid was added, and then lyophilized. This yielded 81 mg (68% of theory) of the title compound.

LC-MS (Method 5): R _t = 2.69 min; MS (EIpos): m/z = 514 [M+H] ⁺ .

Example 198

Trifluoroacetic acid/(2S)-2-amino-N-(2-aminoethyl)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butanamide (1:1)

15 mg (0.018 mmol) of intermediate C64 was dissolved in 4 ml of 2,2,2-trifluoroethanol. 15 ml (0.110 mmol) of zinc chloride was added, and the preparation was stirred at 50°C for 2 hours. 32 mg (0.110 mmol) of ethylenediamine-N,N,N',N'-tetraacetic acid was then added, and the preparation was concentrated under vacuum. The residue was purified by preparative HPLC. Concentration of the appropriate fractions and lyophilization of the residue from acetonitrile/water yielded 9.5 mg (77% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.68 min; MS (ESIpos): m/z = 556 (M+H) ⁺ .

Example 199

4-[(2-{[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butanoyl}amino)ethyl]amino}-2-oxoethyl)amino]-3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-4-oxobutanoic acid/trifluoroacetic acid (1:1)

and

4-[(2-{[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butanoyl}amino)ethyl]amino}-2-oxoethyl)amino]-2-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-4-oxobutanoic acid/trifluoroacetic acid (1:1)

LC-MS (Method 1): R _t = 0.80 min; MS (EIpos): m/z = 814 [M+H] ⁺ .

First, L-cysteine is converted to N -{ [ 2-(trimethylsilyl)ethoxy]carbonyl}-L-cysteine using 1-({[2-(trimethylsilyl)ethoxy]carbonyl}oxy)pyrrolidine-2,5-dione in DMF in the presence of N,N-diisopropylethylamine.

406 mg (1.53 mmol) of N-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-cysteine was dissolved in 10 mL of DMF, 157.5 mg (1.606 mmol) of maleic anhydride was added, and the mixture was stirred at room temperature for 1 hour. To 130 μL of this solution, 7.5 mg (0.01 mmol) of intermediate C66 was added, and the mixture was stirred at room temperature for 5 minutes. The mixture was then concentrated under vacuum, and the residue was purified by preparative HPLC. The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 10 mg (89%) of the protected intermediate; separation of the regioisomers was not possible either by HPLC or by LC-MS.

LC-MS (Method 1): R _t = 1.38 min; MS (EIpos): m/z = 1120 [M+H] ⁺ .

In the final step, 10 mg of this intermediate was dissolved in 2 ml of 2,2,2-trifluoroethanol. 12 ml (0.088 mmol) of zinc chloride was added, and the preparation was stirred at 50°C for 30 minutes. Then, 26 mg (0.088 mmol) of ethylenediamine-N,N,N',N'-tetraacetic acid was added, and the solvent was evaporated under vacuum. The residue was purified by preparative HPLC. Concentration of the appropriate fractions and lyophilization of the residue from acetonitrile/water yielded 8.3 mg (99% of theory) of the title compound as an 87:13 mixture of regioisomers.

LC-MS (Method 5): R _t = 2.3 min and 2.43 min; MS (ESIpos): m/z = 832 (M+H) ⁺ .

¹ H NMR major regioisomers: (500 MHz, DMSO-d ₆ ): d = 8.7 (m, 1H), 8.5 (m,2H), 8.1 (m, 1H), 7.6 (m, 1H), 7.5 (s, 1H) 7.4-7.15 (m, 6H), 6.9-7.0 (m, 1H),6.85 (s, 1H), 5.61 (s, 1H), 4.9 and 5.2 (2d, 2H), 4.26 and 4.06 (2d, 2H), 3.5-3.8 (m, 5H), 3.0-3.4 (m, 5H), 2.75-3.0 (m, 3H), 2.58 and 2.57 (dd, 1H), 0.77 and 1.5 (2m, 2H), 0.81 (s, 9H).

Alternatively, the regioisomeric title compound was prepared as follows:

For this purpose, L-cysteine is first converted into N -{[2-(trimethylsilyl)ethoxy]carbonyl}-L-cysteine in DMF with 1-({[2-(trimethylsilyl)ethoxy]carbonyl}oxy)pyrrolidine-2,5-dione in the presence of N,N-diisopropylethylamine.

55 mg (0.068 mmol) of intermediate F104 and 36 mg (0.136 mmol) of N-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-cysteine were dissolved in 15 ml of DMF and stirred at room temperature for 20 hours. The mixture was then concentrated, and the residue was purified by preparative HPLC. After combining the appropriate fractions and evaporating the solvent under vacuum, the residue was dissolved in 15 ml of THF/water (1:1). 131 μl of 2M aqueous lithium hydroxide solution was added, and the mixture was stirred at room temperature for 1 hour. The reaction was then neutralized with 1M hydrochloric acid, the solvent evaporated under vacuum, and the residue purified by preparative HPLC. This yielded 37 mg (50% of theory) of the regioisomeric protected intermediate as a colorless foam.

LC-MS (Method 5): R _t = 3.33 min and 3.36 min; MS (ESIpos): m/z = 976 (M+H) ⁺ .

In the final step, 25 mg (0.023 mmol) of this intermediate was dissolved in 3 ml of 2,2,2-trifluoroethanol. 12.5 ml (0.092 mmol) of zinc chloride was added, and the preparation was stirred at 50°C for 4 hours. Then, 27 mg (0.092 mmol) of ethylenediamine-N,N,N',N'-tetraacetic acid was added, and the solvent was evaporated under vacuum. The residue was purified by preparative HPLC. Concentration of the appropriate fractions and lyophilization of the residue from acetonitrile/water yielded 18.5 mg (85% of theory) of the title compound as a 21:79 mixture of regioisomers.

LC-MS (Method 5): R _t = 2.37 min and 3.44 min; MS (ESIpos): m/z = 832 (M+H) ⁺ .

The directed preparation of the individual regioisomers of the title compound was carried out as follows.

Example 199-2

4-[(2-{[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butanoyl}amino)ethyl]amino}-2-oxoethyl)amino]-2-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-4-oxobutanoic acid/trifluoroacetic acid (1:1)

For this purpose, L-cysteine methyl ester is first converted into N-{ [2-(trimethylsilyl)ethoxy]carbonyl}-L-cysteine methyl ester in DMF with 1-({[2-(trimethylsilyl)ethoxy]carbonyl}oxy)pyrrolidine-2,5-dione in the presence of N,N-diisopropylethylamine.

53 mg (0.251 mmol) of commercially available 3-bromo-4-methoxy-4-oxobutanoic acid and 70 mg (0.251 mmol) of N-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-cysteine methyl ester were dissolved in 5 ml of DMF, and aqueous sodium bicarbonate was added while monitoring the pH. After stirring at room temperature for 15 minutes, the pH was adjusted to 4.3 with acetic acid, and the preparation was concentrated. The residue was purified by preparative HPLC. Combining the appropriate fractions and evaporating the solvent under vacuum yielded 72 mg (70% of theory) of 4-methoxy-3-{[(2R)-3-methoxy-3-oxo-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)propyl]sulfanyl}-4-oxobutanoic acid.

LC-MS (Method 1): R _t = 0.93 min; MS (ESIpos): m/z = 410 (M+H) ⁺ .

This intermediate is coupled with intermediate C66 in the presence of HATU and then first deprotected with lithium hydroxide in methanol as described above and then with zinc chloride. The residue is purified by preparative HPLC. Concentration of the appropriate fractions and the lyophilization of the residue from acetonitrile/water produce 2 mg of the title compound.

LC-MS (Method 1): R _t = 0.78 min; MS (ESIpos): m/z = 832 (M+H) ⁺ .

Isomer 1 can be prepared in a similar manner.

Example 200

N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butyryl}-β-alanine/trifluoroacetic acid (1:1)

10 mg (0.014 mmol) of intermediate C61 was dissolved in 3 ml of 2,2,2-trifluoroethanol. 11 ml (0.082 mmol) of zinc chloride was added, and the preparation was stirred at 50°C for 30 minutes. Then, 24 mg (0.082 mmol) of ethylenediamine-N,N,N',N'-tetraacetic acid was added, and the solvent was evaporated under vacuum. The residue was purified by preparative HPLC. Concentration of the appropriate fractions and lyophilization of the residue from acetonitrile/water yielded 4.2 mg (40% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.79 min; MS (ESIpos): m/z = 585 (M+H) ⁺ .

Example 201

N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butyryl}-L-serine/trifluoroacetic acid (1:1)

20 mg (0.03 mmol) of intermediate C58 and 8.5 mg (0.037 mmol) of L-serine benzyl ester hydrochloride (1:1) were placed in 5 mL of DMF, and 17 mg (0.046 mmol) of HATU and 21 μL of N , N -diisopropylethylamine were added. After stirring at room temperature for 10 minutes, the mixture was concentrated, and the residue was purified by preparative HPLC. This yielded 12.5 mg (49% of theory) of the intermediate. LC-MS (Method 1): R _t = 1.42 min; MS (ESIpos): m/z = 835 (M+H) ⁺ .

12.5 mg (0.015 mmol) of this intermediate were dissolved in 10 ml of ethanol, palladium on carbon (10%) was added, and the mixture was hydrogenated with hydrogen at standard pressure at room temperature for 30 minutes. The catalyst was filtered off and the solvent was evaporated under vacuum, yielding 7.5 mg (67% of theory) of the intermediate after lyophilization from acetonitrile/water.

LC-MS (Method 1): R _t = 1.28 min; MS (ESIpos): m/z = 745 (M+H) ⁺ .

7.5 mg (0.01 mmol) of this intermediate was dissolved in 3 ml of 2,2,2-trifluoroethanol. 8 ml (0.06 mmol) of zinc chloride was added, and the mixture was stirred at 50°C for 4.5 hours. Then, 17.7 mg (0.06 mmol) of ethylenediamine-N,N,N',N'-tetraacetic acid was added, and the solvent was evaporated under vacuum. The residue was purified by preparative HPLC. Concentration of the appropriate fractions and lyophilization of the residue from acetonitrile/water yielded 4.2 mg (58% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.75 min; MS (ESIpos): m/z = 601 (M+H) ⁺ .

Example 202

N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butyryl}-L-alanine/trifluoroacetic acid (1:1)

The title compound was prepared in analogy to Example 201 from intermediate C58 and L-alanine benzyl ester hydrochloride (1:1).

LC-MS (Method 1): R _t = 0.82 min; MS (ESIpos): m/z = 585 (M+H) ⁺ .

Example 203

N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butyryl}glycine/trifluoroacetic acid (1:1)

The title compound was prepared in analogy to Example 201 from intermediate C58 and glycine benzyl ester hydrochloride (1:1).

LC-MS (Method 1): R _t = 0.82 min; MS (ESIpos): m/z = 571 (M+H) ⁺ .

Example 204A

Here, 5 mg of cetuximab (c = 23.1 mg/ml) in PBS was used for coupling to intermediate F204, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.88 mg/ml

Drug/mAb ratio: 2.6.

Example 204B

Here, 50 mg of anti-TWEAKR AK-1 (c = 18.6 mg/ml) in PBS was used to couple with intermediate F204, and the preparation was concentrated by ultracentrifugation after dextran gel purification, rediluted with PBS, and reconcentrated. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 12.66 mg/ml

Drug/mAb ratio: 3.5.

Example 204E

Here, 5.0 mg of trastuzumab antibody (c = 13.5 mg/ml) in PBS was used for coupling to intermediate F204, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.65 mg/ml

Drug/mAb ratio: 3.5.

Example 205A

Here, 5 mg of cetuximab (c = 23.1 mg/ml) in PBS was used to couple with intermediate F205, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.99 mg/ml

Drug/mAb ratio: 3.2.

Example 205B

Here, 5 mg of anti-TWEAKR AK-1 (c = 18.6 mg/ml) in PBS was used to couple with intermediate F205, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 0.96 mg/ml

Drug/mAb ratio: 2.6.

Example 205E

Here, 5 mg of trastuzumab (c = 13.5 mg/ml) in PBS was used to couple with intermediate F205, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.94 mg/ml

Drug/mAb ratio: 3.6.

Example 206A

Here, 5 mg of cetuximab (c = 23.1 mg/ml) in PBS was used for coupling to intermediate F206, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.73 mg/ml

Drug/mAb ratio: 2.6.

Example 206B

Here, 5 mg of anti-TWEAKR AK-1 (c = 18.6 mg/ml) in PBS was used to couple with intermediate F206, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.21 mg/ml

Drug/mAb ratio: 2.0.

Example 206E

Here, 5 mg of trastuzumab (c = 13.5 mg/ml) in PBS was used for coupling to intermediate F206, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.84 mg/ml

Drug/mAb ratio: 2.8.

Example 207A

Here, 5 mg of cetuximab (c = 10 mg/ml) in PBS was used to couple with intermediate F207, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.79 mg/ml

Drug/mAb ratio: 3.4.

Example 207B

Here, 50 mg of anti-TWEAKR AK-1 (c = 18.6 mg/ml) in PBS was conjugated to intermediate F207. After purification on a dextran gel, the preparation was concentrated by ultracentrifugation, rediluted with PBS, and reconcentrated. Some ADC may also exist as hydrolyzed open-chain succinamide attached to the antibody. For this ADC preparation, a 19% content of the open-ring succinamide form was measured immediately after synthesis.

Protein concentration: 12.99 mg/ml

Drug/mAb ratio: 4.3.

Example 207E

Here, 5 mg of trastuzumab (c = 13.5 mg/ml) in PBS was used to couple with intermediate F207, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.39 mg/ml

Drug/mAb ratio: 2.8.

Example 207I

Here, 5.0 mg of nimotuzumab (c = 13.1 mg/ml) in PBS was used for coupling to intermediate F207, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted with PBS. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.92 mg/ml

Drug/mAb ratio: 3.5.

Example 207H

Here, 5.0 mg of panitumumab (c = 13.6 mg/ml) in PBS was coupled to intermediate F207. The reduction time with TCEP was 4 hours, and the stirring time for ADC coupling was 20 hours. After purification on a dextran gel, the formulation was concentrated by ultracentrifugation and rediluted with PBS. Some ADC may also be present as hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.0 mg/ml

Drug/mAb ratio: 1.9.

Example 208A

Under argon, a solution of 0.344 mg of TCEP in 100 μL of PBS buffer was added to 60 mg of cetuximab (c = 10.92 mg/ml) in 5494 μL of PBS. The formulation was stirred at room temperature for 30 minutes, followed by the addition of 2.582 mg (0.003 mmol) of Intermediate F104 dissolved in 600 μL of DMSO. After stirring at room temperature for an additional 120 minutes, the formulation was diluted with 1306 μL of PBS buffer pre-adjusted to pH 8.

This solution was then applied to a PD 10 column ( ^Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer, pH 8, and eluted with PBS buffer, pH 8. The eluate was diluted to a total volume of 14 ml with PBS buffer, pH 8. This solution was stirred at room temperature under argon overnight, then concentrated by ultracentrifugation, rediluted with PBS buffer (pH 7.2), and reconcentrated. The resulting ADC batch was characterized as follows:

Protein concentration: 13.36 mg/ml

Drug/mAb ratio: 1.8.

For this ADC preparation, a content of 94% of the open-ring succinamide form was determined.

Example 208B

Under argon, 60 mg of anti-TWEAKR AK-1 (c = 18.6 mg/ml) in 3225 μl of PBS was diluted with 775 μl of PBS buffer, and then a solution of 0.344 mg of TCEP in 100 μl of PBS buffer was added. The preparation was stirred at room temperature for 30 minutes, and then 2.582 mg (0.003 mmol) of intermediate F104 dissolved in 600 μl of DMSO was added. After stirring at room temperature for an additional 120 minutes, the preparation was diluted with 300 μl of PBS buffer pre-adjusted to pH 8.

This solution was then applied to a PD 10 column ( ^Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer, pH 8, and eluted with PBS buffer, pH 8. The eluate was diluted to a total volume of 14 ml with PBS buffer, pH 8. This solution was stirred at room temperature under argon overnight, then concentrated by ultracentrifugation, rediluted with PBS buffer (pH 7.2), and reconcentrated. The resulting ADC batch was characterized as follows:

Protein concentration: 14.95 mg/ml

Drug/mAb ratio: 3.2.

Example 208I

Under argon, a solution of 0.344 mg of TCEP in 100 μL of PBS buffer was added to 60 mg of nimotuzumab (c = 13.1 mg/mL) in 4587 μL of PBS. The formulation was stirred at room temperature for 30 minutes, followed by the addition of 2.582 mg (0.003 mmol) of Intermediate F104 dissolved in 600 μL of DMSO. After stirring at room temperature for an additional 120 minutes, the reaction was diluted with 2213 μL of PBS buffer pre-adjusted to pH 8.

This solution was then applied to a PD 10 column ( ^Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer, pH 8, and eluted with PBS buffer, pH 8. The eluate was diluted to a total volume of 14 ml with PBS buffer, pH 8. This solution was stirred at room temperature under argon overnight, then concentrated by ultracentrifugation, rediluted with PBS buffer (pH 7.2), and reconcentrated. The resulting ADC batch was characterized as follows:

Protein concentration: 14.79 mg/ml

Drug/mAb ratio: 3.1.

For this ADC preparation, a content of 91% of the open-ring succinamide form was determined.

Example 208K

Under argon, a solution of 0.23 mg of TCEP in 67 μl of PBS buffer was added to 40 mg of anti-TWEAKR AK-2 (c = 14.5 mg/ml) in 2759 μl of PBS. The preparation was stirred at room temperature for 30 minutes, and then 1.72 mg (0.002 mmol) of intermediate F104 dissolved in 400 μl of DMSO was added. After stirring at room temperature for another 120 minutes, the preparation was diluted with 1774 μl of PBS buffer pre-adjusted to pH 8.

This solution was then applied to a PD 10 column ( ^Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer, pH 8, and eluted with PBS buffer, pH 8. The eluate was diluted to a total volume of 14 ml with PBS buffer, pH 8. This solution was stirred at room temperature under argon overnight, then concentrated by ultracentrifugation, rediluted with PBS buffer (pH 7.2), and reconcentrated. The resulting ADC batch was characterized as follows:

Protein concentration: 11.66 mg/ml

Drug/mAb ratio: 3.1.

Example 209A

Here, 5.0 mg of cetuximab (c = 21.32 mg/ml) in PBS was used to couple to intermediate F209, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.75 mg/ml

Drug/mAb ratio: 2.4.

Example 209B

Here, 5.0 mg of anti-TWEAKR AK-1 antibody (c = 18.60 mg/ml) in PBS was conjugated to intermediate F209, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.30 mg/ml

Drug/mAb ratio: 2.1.

Example 209E

Here, 5.0 mg of trastuzumab antibody (c = 13.50 mg/ml) in PBS was conjugated to intermediate F209, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.03 mg/ml

Drug/mAb ratio: 2.3.

Example 209H

Here, 5.0 mg of panitumumab antibody (c = 70.5 mg/ml) in PBS was conjugated to intermediate F209, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.91 mg/ml

Drug/mAb ratio: 1.5.

Example 209I

Here, 5.0 mg of nimotuzumab antibody (c = 13.1 mg/ml) in PBS was conjugated to intermediate F209, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.81 mg/ml

Drug/mAb ratio: 2.5.

Example 210A

Here, 5.0 mg of cetuximab (c = 21.32 mg/ml) in PBS was used for conjugation with intermediate F210, and the formulation was concentrated by ultracentrifugation after purification on a dextran gel and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.92 mg/ml

Drug/mAb ratio: 2.9.

Example 210B

Here, 5.0 mg of anti-TWEAKR AK-1 antibody (c = 18.60 mg/ml) in PBS was conjugated to intermediate F210, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.41 mg/ml

Drug/mAb ratio: 2.5.

Example 210E

Here, 5.0 mg of trastuzumab antibody (c = 13.50 mg/ml) in PBS was conjugated to intermediate F210, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.79 mg/ml

Drug/mAb ratio: 2.8.

Example 211A

Here, 5.0 mg of cetuximab (c = 12.33 mg/ml) in PBS was used for conjugation with intermediate F211, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.82 mg/ml

Drug/mAb ratio: 2.0.

Example 211B

Here, 5.0 mg of anti-TWEAKR AK-1 antibody (c = 34.42 mg/ml) in PBS was conjugated to intermediate F211, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.52 mg/ml

Drug/mAb ratio: 2.4.

Example 211E

Here, 5.0 mg of trastuzumab antibody (c = 13.50 mg/ml) in PBS was conjugated to intermediate F211, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.84 mg/ml

Drug/mAb ratio: 2.4.

Example 212A

Here, 5.0 mg of cetuximab (c = 11.3 mg/ml) in PBS was used for conjugation with intermediate F212, and the formulation was concentrated by ultracentrifugation after purification on a dextran gel and rediluted. Some of the ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.94 mg/ml

Drug/mAb ratio: 3.3.

Example 212B

Here, 5.0 mg of anti-TWEAKR AK-1 antibody (c = 18.6 mg/ml) in PBS was conjugated to intermediate F212, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 0.85 mg/ml

Drug/mAb ratio: 2.0.

Example 212E

Here, 5.0 mg of trastuzumab antibody (c = 13.50 mg/ml) in PBS was conjugated to intermediate F212, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.55 mg/ml

Drug/mAb ratio: 2.0.

Example 213A

Here, 5.0 mg of cetuximab (c = 21.32 mg/ml) in PBS was used for conjugation with intermediate F213, and the formulation was concentrated by ultracentrifugation after purification on a dextran gel and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.83 mg/ml

Drug/mAb ratio: 2.4.

Example 213B

Here, 5.0 mg of anti-TWEAKR AK-1 antibody (c = 18.6 mg/ml) in PBS was conjugated to intermediate F213, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.4 mg/ml

Drug/mAb ratio: 2.3.

Example 213E

Here, 5.0 mg of trastuzumab antibody (c = 13.50 mg/ml) in PBS was conjugated to intermediate F213, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.94 mg/ml

Drug/mAb ratio: 2.5.

Example 214A

Here, 5.0 mg of cetuximab (c = 15.21 mg/ml) in PBS was used for conjugation with intermediate F214, and the formulation was concentrated by ultracentrifugation after purification on a dextran gel and rediluted. Some of the ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.00 mg/ml

Drug/mAb ratio: 2.6.

Example 214B

Here, 5.0 mg of anti-TWEAKR AK-1 antibody (c = 18.6 mg/ml) in PBS was conjugated to intermediate F214, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.01 mg/ml

Drug/mAb ratio: 2.6.

Example 214E

Here, 5.0 mg of trastuzumab antibody (c = 13.50 mg/ml) in PBS was conjugated to intermediate F214, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.86 mg/ml

Drug/mAb ratio: 2.7.

Example 215A

Here, 5.0 mg of cetuximab (c = 15.21 mg/ml) in PBS was used for conjugation with intermediate F215, and the formulation was concentrated by ultracentrifugation after purification on a dextran gel and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.99 mg/ml

Drug/mAb ratio: 2.7.

Example 215B

Here, 5.0 mg of anti-TWEAKR AK-1 antibody (c = 18.6 mg/ml) in PBS was conjugated to intermediate F215, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.64 mg/ml

Drug/mAb ratio: 2.8.

Example 215E

Here, 5.0 mg of trastuzumab antibody (c = 13.50 mg/ml) in PBS was conjugated to intermediate F215, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.84 mg/ml

Drug/mAb ratio: 2.8.

Example 215H

Here, 5.0 mg of panitumumab antibody (c = 70.5 mg/ml) in PBS was conjugated to intermediate F215, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.86 mg/ml

Drug/mAb ratio: 1.4.

Example 215I

Here, 5.0 mg of nimotuzumab (c = 13.1 mg/ml) in PBS was used for conjugation with intermediate F215, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.83 mg/ml

Drug/mAb ratio: 2.6.

Example 216A

Here, 5.0 mg of cetuximab (c = 15.21 mg/ml) in PBS was used for conjugation with intermediate F216, and the formulation was concentrated by ultracentrifugation after purification on a dextran gel and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.97 mg/ml

Drug/mAb ratio: 2.8.

Example 216B

Here, 5.0 mg of anti-TWEAKR AK-1 antibody (c = 18.6 mg/ml) in PBS was conjugated to intermediate F216, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.94 mg/ml

Drug/mAb ratio: 2.5.

Example 216E

Here, 5.0 mg of trastuzumab antibody (c = 13.50 mg/ml) in PBS was conjugated to intermediate F216, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.90 mg/ml

Drug/mAb ratio: 1.7.

Example 217A

Here, 5.0 mg of cetuximab (c = 15.21 mg/ml) in PBS was used for conjugation with intermediate F217, and the formulation was concentrated by ultracentrifugation after purification on a dextran gel and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.05 mg/ml

Drug/mAb ratio: 2.7.

Example 217B

Here, 5.0 mg of anti-TWEAKR AK-1 antibody (c = 18.6 mg/ml) in PBS was conjugated to intermediate F217, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.44 mg/ml

Drug/mAb ratio: 2.4.

Example 217E

Here, 5.0 mg of trastuzumab antibody (c = 13.50 mg/ml) in PBS was conjugated to intermediate F217, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.85 mg/ml

Drug/mAb ratio: 2.8.

Example 218A

Here, 5.0 mg of cetuximab (c = 15.21 mg/ml) in PBS was used for conjugation with intermediate F218, and the formulation was concentrated by ultracentrifugation after purification on a dextran gel and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.05 mg/ml

Drug/mAb ratio: 3.0.

Example 218B

Here, 5.0 mg of anti-TWEAKR AK-1 antibody (c = 18.6 mg/ml) in PBS was conjugated to intermediate F218, and the preparation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.95 mg/ml

Drug/mAb ratio: 2.9.

Example 218E

Here, 5.0 mg of trastuzumab antibody (c = 13.50 mg/ml) in PBS was conjugated to intermediate F218, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.84 mg/ml

Drug/mAb ratio: 2.7.

Example 218H

Here, 5.0 mg of panitumumab antibody (c = 20 mg/ml) in PBS was conjugated to intermediate F218. The reduction time with TCEP was increased to 4 hours, and the stirring time for ADC conjugation was increased to 20 hours. After purification on a dextran gel, the reaction was concentrated by ultracentrifugation and rediluted. Some ADC may also be present as hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.33 mg/ml

Drug/mAb ratio: 0.8.

Example 218I

Here, 5.0 mg of nimotuzumab antibody (c = 13.8 mg/ml) in PBS was conjugated to intermediate F218, and the formulation was concentrated by ultracentrifugation after dextran gel purification and rediluted. Some ADC may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.48 mg/ml

Drug/mAb ratio: 3.0.

Example 219

Trifluoroacetic acid/N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-4-methylbenzamide (1:1)

70.0 mg (0.09 mmol) of 9H-fluoren-9-ylmethyl [3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)propyl]carbamate (Intermediate C67) was initially charged in 3.0 mL of dichloromethane, and 31.3 mg (0.31 mmol) of triethylamine and 31.8 mg (0.21 mmol) of 4-methylbenzoyl chloride were added. The reaction mixture was stirred at room temperature overnight. The solvent was evaporated under vacuum, and the residue was directly purified by preparative RP-HPLC (column: Reprosil 125x30; 10µm, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 33.4 mg (48% of theory) of the compound 9H-fluoren-9-ylmethyl {3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(4-methylbenzoyl)amino]propyl}carbamate.

LC-MS (Method 2): R _t = 11.91 min; MS (ESIpos): m/z = 774 (M+Na) ⁺ .

33.0 mg (0.04 mmol) of 9H-fluoren-9-ylmethyl {3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(4-methylbenzoyl)amino]propyl}carbamate was stirred overnight with 20.0 mg (0.23 mmol) of morpholine in 1.0 ml of DMF. The reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 125x30; 10µm, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 9.6 mg (34% of theory) of the title compound.

LC-MS (Method 1): R _t = 1.01 min; MS (ESIpos): m/z = 530 (M+H) ⁺ .

Example 220

Trifluoroacetic acid/N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-4-(methylsulfanyl)benzamide (1:1)

Initially, 50.0 mg (0.07 mmol) of 9H-fluoren-9-ylmethyl [3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)propyl]carbamate (Intermediate C67) was charged in 2.0 ml of dichloromethane and 22.3 mg (0.22 mmol) of triethylamine and 27.5 mg (0.15 mmol) of 4-(methylsulfanyl)benzoyl chloride were added. The reaction mixture was stirred at 40° C. for 4 hours, and another 10.2 mg (0.10 mmol) of triethylamine and 27.5 mg (0.15 mmol) of 4-(methylsulfanyl)benzoyl chloride were added and stirred at room temperature overnight. An additional 14.9 mg (0.15 mmol) of triethylamine and 27.5 mg (0.15 mmol) of 4-(methylsulfanyl)benzoyl chloride were then added, and the mixture was stirred at 40°C for 2 hours. The mixture was diluted with ethyl acetate, and the organic phase was washed three times with water and once with saturated NaCl solution. The organic phase was dried over magnesium sulfate and concentrated under vacuum. The residue was purified by preparative RP-HPLC (column: Reprosil 125x30; 10µm, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 39.8 mg (76% of theory) of the compound [9H-fluoren-9-ylmethyl 3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}[4-(methylsulfanyl)benzoyl]amino)propyl]carbamate].

LC-MS (Method 1): R _t = 1.59 min; MS (ESIpos): m/z = 785 (M+H) ⁺ .

18.0 mg (0.02 mmol) of 9H-fluoren-9-ylmethyl [3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}[4-(methylsulfanyl)benzoyl]amino)propyl]carbamate were stirred overnight with 10.0 mg (0.12 mmol) of morpholine in 1.0 ml of DMF. The reaction mixture was purified directly by preparative RP-HPLC (column: Reprosil 125x30; 10 µm, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 6.2 mg (40% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.99 min; MS (ESIpos): m/z = 562 (M+H) ⁺ .

Example 221

Trifluoroacetic acid/(2S)-N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2-hydroxypropionamide (1:1)

40.0 mg (0.06 mmol) of 9H-fluoren-9-ylmethyl [3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)propyl]carbamate (Intermediate C67) was initially charged in 2.0 ml of dichloromethane, and 9.6 mg (0.10 mmol) of triethylamine and 14.3 mg (0.10 mmol) of (2S)-1-chloro-1-oxopropan-2-yl acetate were added. The reaction mixture was stirred at room temperature overnight. The solvent was evaporated under vacuum, and the residue was purified by preparative RP-HPLC (column: Reprosil 125x30; 10µm, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 39.7 mg (84% of theory) of the compound (2S)-1-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(3-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}propyl)amino]-1-oxopropan-2-yl acetate.

LC-MS (Method 1): R _t = 1.51 min; MS (ESIpos): m/z = 748 (M+H) ⁺ .

37.0 mg (0.05 mmol) of (2S)-1-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(3-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}propyl)amino]-1-oxopropane-2-acetate was stirred in 1.0 ml of DMF with 0.1 ml of morpholine and 3 drops of water at 50°C for 10 hours. An additional 0.1 ml of morpholine and 0.1 ml of water were added, and the mixture was stirred at 50°C for 10 hours. After adding 20.5 mg (0.15 mmol) of potassium carbonate and stirring at room temperature for 72 hours, 0.1 ml of 1N NaOH solution was added, and the mixture was stirred at room temperature overnight. The reaction mixture was purified directly by preparative RP-HPLC (column: Reprosil 125x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 20.5 mg (69% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.89 min; MS (ESIpos): m/z = 484 (M+H) ⁺ .

Example 222

Trifluoroacetic acid/N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2-(methylsulfanyl)acetamide (1:1)

Initially, 70.0 mg (0.11 mmol) of 2-(trimethylsilyl)ethyl 3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(chloroacetyl)amino]propyl}carbamate (Intermediate C70) was charged in 3.0 mL of DMF. 15.5 mg (0.22 mmol) of sodium methanethiolate was added, and the reaction mixture was stirred at 50°C for 2 hours. The reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 125x30; 10µm, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 60.0 mg (84% of theory) of the compound 2-(trimethylsilyl)ethyl 3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}[(methylsulfanyl)acetyl]amino)propyl]carbamate.

LC-MS (Method 1): R _t = 1.50 min; MS (ESIpos): m/z = 644 (M+H) ⁺ .

40.0 mg (0.06 mol) of 2-(trimethylsilyl)ethyl 3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}[(methylsulfanyl)acetyl]amino)propyl]carbamate was dissolved in 2.0 ml of trifluoroethanol, and 21.2 mg (0.16 mmol) of zinc dichloride was added. The reaction mixture was stirred at 50°C overnight. 45.4 mg (0.01 mmol) of ethylenediamine-N,N,N',N'-tetraacetic acid was added, stirred for 10 minutes, and then water (0.1% TFA) was added. Purification was carried out directly by preparative RP-HPLC (column: Reprosil 125x30; 10µm, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 34.6 mg (91% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.92 min; MS (ESIpos): m/z = 500 (M+H) ⁺ .

Example 223

Trifluoroacetic acid/(2S)-N-(3-aminopropyl)-N-{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}-2-hydroxypropionamide (1:1)

40.0 mg (0.08 mmol) of tert-butyl [3-({(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}amino)propyl]carbamate was dissolved in 2.0 ml of dichloromethane, and 19.7 mg (0.20 mmol) of triethylamine and 29.4 mg (0.20 mmol) of (2S)-1-chloro-1-oxopropan-2-yl acetate were added. The reaction mixture was stirred overnight. The solvent was evaporated under vacuum, and the residue was purified by preparative RP-HPLC (column: Reprosil 125x30; 10µm, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This gave 21.2 mg (43% of theory) of the compound (2S)-1-({(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}{3-[(tert-butoxycarbonyl)amino]propyl}amino)-1-oxopropan-2-yl acetate.

LC-MS (Method 1): R _t = 1.46 min; MS (ESIpos): m/z = 627 (M+H) ⁺ .

21.2 mg (0.03 mmol) of (2S)-1-({(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}{3-[(tert-butoxycarbonyl)amino]propyl}amino)-1-oxopropane-2-acetate were dissolved in 1.0 ml of dichloromethane, 77.1 mg (0.68 mmol) of trifluoroacetic acid were added, and the mixture was stirred at room temperature for 2 hours. Two additional 77.1 mg (0.68 mmol) of trifluoroacetic acid were added, and the mixture was stirred at room temperature overnight each time. The solvent was evaporated under vacuum, and the residue was repeatedly co-distilled with dichloromethane and then dried under high vacuum. The residue, which comprises trifluoroacetic acid/(2S)-1-[(3-aminopropyl){(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}amino]-1-oxopropan-2-yl acetate (1:1), is reacted further without further purification.

LC-MS (Method 1): R _t = 0.92 min; MS (ESIpos): m/z = 527 (M+H) ⁺ .

26.5 mg (0.04 mmol) of (2S)-1-[(3-aminopropyl){(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}amino]-1-oxopropan-2-yl acetate (1:1) was dissolved in THF/methanol/water (1.0 ml/1.0 ml/0.05 ml), and 17.2 mg of potassium carbonate was added. The reaction mixture was stirred at room temperature overnight. The reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 125x30; 10µm, flow rate: 50 ml/min, MeCN/water; 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 17.3 mg (70% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.91 min; MS (ESIpos): m/z = 485 (M+H) ⁺ .

Example 224

Trifluoroacetic acid/methyl 4-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-4-oxobutanoate (1:1)

60.0 mg (0.11 mmol) of 2-(trimethylsilyl)ethyl [3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)propyl]carbamate (see Synthesis of Intermediate C11) was dissolved in 1.0 ml of dichloromethane, and 19.6 mg (0.25 mmol) of pyridine and 35.8 mg (0.24 mmol) of methyl 4-chloro-4-oxobutanoate were added. The reaction mixture was stirred at 40°C overnight. An additional 19.6 mg (0.25 mmol) of pyridine and 35.8 mg (0.24 mmol) of methyl 4-chloro-4-oxobutanoate were added, and the mixture was stirred at 40°C overnight. The solvent was evaporated under vacuum, and the residue was purified by preparative RP-HPLC (column: Reprosil 125x30; 10µm, flow rate: 50 ml/min, MeCN/water). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 16.1 mg (22% of theory) of the compound 11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silapentadecan-15-oic acid methyl ester.

LC-MS (Method 1): R _t = 1.52 min; MS (ESIpos): m/z = 670 (M+H) ⁺ .

Dissolve 16.1 mg (0.02 mmol) of methyl 11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silapentadecan-15-oate in 1.0 ml of trifluoroethanol, and add 16.4 mg (0.12 mmol) of zinc dichloride. The reaction mixture is stirred at 50°C for 5 hours. Then, 35.1 mg (0.12 mmol) of ethylenediamine-N,N,N',N'-tetraacetic acid is added to the reaction mixture, which is stirred for 10 minutes, followed by the addition of water (0.1% TFA). Purify directly by preparative RP-HPLC (column: Reprosil 125x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent is evaporated under vacuum, and the residue is dried under high vacuum. This yields 11.1 mg (72% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.93 min; MS (ESIpos): m/z = 526 (M+H) ⁺ .

Example 225

4-[(3-Aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-4-oxobutanoic acid/trifluoroacetic acid (1:1)

9.7 mg (0.02 mmol) of trifluoroacetic acid/methyl 4-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-4-oxobutanoate (1:1) (Example 224) were initially charged in THF/methanol/water (1.0 ml/0.2 ml/0.04 ml), and 1.3 mg (0.03 mmol) of lithium hydroxide monohydrate was added. The reaction mixture was stirred at room temperature overnight. An additional 1.3 mg (0.03 mmol) of lithium hydroxide monohydrate was added and stirred at room temperature overnight. 3.6 mg (0.06 mmol) of HOAc was added, and the reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 125x30; 10µm, flow rate: 50 ml/min, MeCN/water; 0.1% TFA). The solvent was evaporated under vacuum and the residue was dried under high vacuum. This gave 5.4 mg (57% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.90 min; MS (ESIpos): m/z = 512 (M+H) ⁺ .

Example 226

(2R)-22-[(3R/S)-3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl]-2-[({2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}sulfanyl)methyl]-4,20-dioxo-7,10,13,16-tetraoxa-3,19-diazadocosan-1-oic acid/trifluoroacetic acid (1:2)

10.3 mg (mmol) of R/S-{2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}-N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecanoyl]-L-cysteine/trifluoroacetic acid (1:1) (Intermediate F209) was initially charged in DMF/water (2.0 ml/0.2 ml), L-cysteine was added, and the mixture was stirred at room temperature for 10 minutes. Water was added to the reaction mixture and purified directly by preparative RP-HPLC (column: Reprosil 125x30; 10µm, flow rate: 50 ml/min, MeCN/water; 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 10.3 mg (82% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.84 min; MS (ESIpos): m/z = 1092 (M+H) ⁺ .

Example 227

Trifluoroacetic acid/4-amino-N-(3-aminopropyl)-N-{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}benzamide (2:1)

50.0 mg (0.10 mol) of tert-butyl [3-({(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}amino)propyl]carbamate (Intermediate C68) was initially charged in dichloromethane and 54.9 mg (0.22 mmol) of tert-butyl [4-(chlorocarbonyl)phenyl]carbamate (Intermediate L71) and 22.7 mg (0.22 mmol) of triethylamine were added. The reaction mixture was stirred at room temperature overnight, and another 54.9 mg (0.22 mmol) of tert-butyl [4-(chlorocarbonyl)phenyl]carbamate (Intermediate L71) and 22.7 mg (0.22 mmol) of triethylamine were added. The reaction mixture was stirred at room temperature overnight. The solvent was evaporated under vacuum, and the residue was purified by preparative RP-HPLC (column: Reprosil 250x40; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 26.2 mg (37% of theory) of the compound tert-butyl [3-({(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}{4-[(tert-butoxycarbonyl)amino]benzoyl}amino)propyl]carbamate.

LC-MS (Method 1): R _t = 5.34 min; MS (ESIpos): m/z = 732 (M+H) ⁺ .

26.2 mg (0.04 mmol) of tert-butyl [3-({(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}{4-[(tert-butoxycarbonyl)amino]benzoyl}amino)propyl]carbamate was dissolved in 2.0 ml of dichloromethane, and 204.1 mg (1.79 mmol) of TFA was added. The reaction mixture was stirred at room temperature overnight. The solvent was evaporated under vacuum, and the residue was purified by preparative RP-HPLC (column: Reprosil 125x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 3.4 mg (13% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.92 min; MS (ESIpos): m/z = 532 (M+H) ⁺ .

Example 228

N-Acetyl-S-{2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}-L-cysteine/trifluoroacetic acid (1:1)

50.0 mg (0.08 mol) of 2-(trimethylsilyl)ethyl 3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(chloroacetyl)amino]propyl}carbamate (Intermediate C70) was suspended in 0.30 mL of water along with 66.43 mg of sodium bicarbonate. A solution of 144.47 g (0.95 mmol) of 1,8-diazabicyclo(5.4.0)undec-7-ene and 51.62 mg (0.32 mmol) of N-acetyl-L-cysteine in 3.0 mL of isopropanol was added to this suspension. The reaction mixture was stirred at 50°C for 2.5 hours. Water (0.1% TFA) was added to the reaction mixture and purified directly by preparative RP-HPLC (column: Reprosil 250x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 55.2 mg (92% of theory) of the compound N-acetyl-S-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl)-L-cysteine.

LC-MS (Method 1): R _t = 1.41 min; MS (ESIpos): m/z = 759 (M+H) ⁺ .

53.1 mg (69.96 μmol) of N-acetyl-S-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl)-L-cysteine was dissolved in 5.0 mL of trifluoroethanol, and 57.2 mg (419.76 μmol) of zinc dichloride was added. The reaction mixture was stirred at 50°C for 4 hours. 122.67 mg (0.42 mmol) of ethylenediamine-N,N,N',N'-tetraacetic acid was added to the reaction mixture, which was stirred for 10 minutes, followed by the addition of water (0.1% TFA). Purification was carried out directly by preparative RP-HPLC (column: Reprosil 125x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). This gave 32.5 mg (64% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.90 min; MS (ESIpos): m/z = 615 (M+H) ⁺ .

Example 229

N-Acetyl-S-[2-([3-(L-alanylamino)propyl]{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)-2-oxoethyl]-L-cysteine/trifluoroacetic acid (1:1)

30.3 mg (41.58 μmol) of N-acetyl-S-{2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}-L-cysteine/trifluoroacetic acid (1:1) (Example 228) was dissolved in 1.5 ml of DMF, and 8.4 mg (83.15 μmol) of 4-methylmorpholine and 14.65 mg (45.73 μmol) of N-[(benzyloxy)carbonyl]-L-alanine 2,5-dioxopyrrolidin-1-yl ester were added. The reaction mixture was stirred at room temperature overnight, and then an additional 8.4 mg (83.15 μmol) of 4-methylmorpholine was added. The reaction mixture was then stirred at room temperature overnight. 10.0 mg (0.17 mmol) of acetic acid was added, and the reaction mixture was purified directly by preparative RP-HPLC (column: Reprosil 250x30; 10µm, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 31.2 mg (92% of theory) of the compound N-acetyl-S-[2-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}[3-({N-[(benzyloxy)carbonyl]-L-alanyl}amino)propyl]amino)-2-oxoethyl]-L-cysteine.

LC-MS (Method 1): R _t = 1.22 min; MS (ESIpos): m/z = 820 (M+H) ⁺ .

28.6 mg (0.04 mmol) of N-acetyl-S-[2-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}[3-({N-[(benzyloxy)carbonyl]-L-alanyl}amino)propyl]amino)-2-oxoethyl]-L-cysteine were dissolved in 5.0 ml of ethanol, and 2.9 mg of palladium-activated carbon (10%) were added. The reaction mixture was hydrogenated overnight at standard pressure and room temperature. The preparation was filtered through Celite, and the filter cake was washed with an ethanol mixture. The solvent was evaporated under vacuum, and the residue was purified by preparative RP-HPLC (column: Reprosil 250x30; 10µm, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was lyophilized. This gave 17.4 mg (62% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.85 min; MS (ESIpos): m/z = 686 (M+H) ⁺ .

Example 230

Trifluoroacetic acid/(2S)-N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}tetrahydrofuran-2-carboxamide (1:1)

100.0 mg (0.16 mmol) of 9H-fluoren-9-ylmethyl [3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)propyl]carbamate (Intermediate C67) was dissolved in 5.0 ml of dichloromethane along with 71.9 mg (0.71 mmol) of triethylamine and added dropwise to a freshly prepared solution of (2S)-tetrahydrofuran-2-carbonyl chloride (preparation: 54.7 mg (0.39 mmol) of (2S)-tetrahydrofuran-2-carboxylic acid was initially charged in 0.7 ml of toluene, 0.04 ml of thionyl chloride was added and stirred at 90° C. for 1 hour. After cooling, the crude reaction solution was reacted further). The reaction mixture was stirred at room temperature overnight. The solvent was evaporated under vacuum, and the residue was purified by preparative RP-HPLC (column: Reprosil 125x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 44.3 mg (38% of theory) of the compound [9H-fluoren-9-ylmethyl 3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}[(2S)-tetrahydrofuran-2-ylcarbonyl]amino)propyl]carbamate].

LC-MS (Method 1): R _t = 1.52 min; MS (ESIpos): m/z = 776 (M+HCOOH-H) ^- .

26.0 mg (0.04 mmol) of 9H-fluoren-9-ylmethyl [3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}[(2S)-tetrahydrofuran-2-ylcarbonyl]amino)propyl]carbamate were dissolved in 2.6 ml of DMF, and 0.26 ml of morpholine was added. The reaction mixture was stirred at room temperature for 3 hours. Purification was carried out directly by preparative RP-HPLC (column: Reprosil 125x30; 10µm, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). This gave 11.7 mg (53% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.93 min; MS (ESIpos): m/z = 510 (M+H) ⁺ .

Example 231

3-({2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}sulfanyl)propanoic acid/trifluoroacetic acid (1:1)

53.9 mg (0.08 mmol) of 11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-14-thia-7,11-diaza-2-silaheptadecane-17-oic acid (Intermediate C69) was dissolved in 4.0 mL of trifluoroethanol, and 31.4 mg (0.23 mmol) of zinc dichloride was added. The reaction mixture was stirred at 50°C overnight. 67.3 mg (0.23 mmol) of ethylenediamine-N,N,N',N'-tetraacetic acid was added to the reaction mixture, which was stirred for 10 minutes, followed by the addition of water (0.1% TFA). Purification was carried out directly by preparative RP-HPLC (column: Reprosil 125x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). This gave 34.2 mg (66% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.91 min; MS (ESIpos): m/z = 558 (M+H) ⁺ .

Example 232

S-{2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}homocysteine/trifluoroacetic acid (1:2)

40.0 mg (47.3 μmol) of S-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridec-13-yl)homocysteine (Intermediate C11) was dissolved in 3.0 mL of trifluoroethanol, and 38.7 mg (0.28 μmol) of zinc dichloride was added. The reaction mixture was stirred at 50°C for 6 days. 83 mg (0.28 mmol) of ethylenediamine-N,N,N',N'-tetraacetic acid was added to the reaction mixture, which was stirred for 10 minutes, followed by the addition of water (0.1% TFA). Purification was carried out directly by preparative RP-HPLC (column: Reprosil 125x30; 10µ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). This gave 32.4 mg (84% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.81 min; MS (ESIpos): m/z = 587 (M+H) ⁺ .

Example 233

Trifluoroacetic acid/4-amino-N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}benzamide (2:1)

73.0 mg (0.12 mmol) of 9H-fluoren-9-ylmethyl [3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)propyl]carbamate (Intermediate C67) and 27.8 mg (0.15 mmol) of 4-nitrobenzoyl chloride were dissolved in 2.0 ml of dichloromethane, and 15.2 mg (0.15 mmol) of triethylamine were added. The reaction mixture was stirred at room temperature overnight. The same amounts of 4-nitrobenzoyl chloride and triethylamine were then added, and the reaction mixture was stirred at room temperature overnight. The solvent was evaporated under vacuum, and the residue was purified by preparative RP-HPLC (column: Reprosil 125x30; 10µm, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). This gave 39.3 mg (44% of theory) of the compound 9H-fluoren-9-ylmethyl {3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(4-nitrobenzoyl)amino]propyl}carbamate.

LC-MS (Method 1): R _t = 1.54 min; MS (ESIpos): m/z = 783 (M+H) ⁺ .

39.3 mg (0.05 mmol) of fluoren-9-ylmethyl {3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(4-nitrobenzoyl)amino]propyl}carbamate was dissolved in 2.0 ml of ethanol, 3.9 mg of palladium hydroxide/activated carbon (20%) was added, and hydrogenation was carried out under standard pressure overnight. The reaction mixture was filtered through a paper filter, and the filter cake was washed with ethanol. The solvent was evaporated under vacuum. The residue was purified by preparative RP-HPLC (column: Reprosil 125x30; 10µm, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 32.9 mg (86% of theory) of the title compound. LC-MS (Method 1): R _t =0.93 min; MS (ESIpos): m/z = 531 (M+H) ⁺ .

Example 234A

Under argon, a solution of 0.029 mg of TCEP in 50 μL of PBS buffer was added to 5 mg of cetuximab (c = 10 mg/ml) in 500 μL of PBS. The formulation was stirred at room temperature for 30 minutes, followed by the addition of 0.2 mg (0.00027 mmol) of intermediate F85 dissolved in 50 μL of DMSO. After stirring at room temperature for an additional 90 minutes, the formulation was diluted with 1900 μL of PBS buffer pre-adjusted to pH 8.

This solution was then applied to a PD 10 column ( ^Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer, pH 8, and eluted with PBS buffer, pH 8. The eluate was stirred overnight at room temperature under argon, then concentrated by ultracentrifugation and rediluted with PBS buffer, pH 7.2. Under these conditions, some ADCs may also exist in a closed-ring form. The resulting ADC batch was characterized as follows:

Protein concentration: 1.68 mg/ml

Drug/mAb ratio: 3.8.

Example 234B

Under argon, a solution of 0.029 mg of TCEP in 50 μl of PBS buffer was added to 5 mg of anti-TWEAKR AK-1 (c = 18.6 mg/ml) in 269 μl of PBS. The preparation was stirred at room temperature for 30 minutes, and then 0.2 mg (0.00027 mmol) of intermediate F85 dissolved in 50 μl of DMSO was added. After stirring at room temperature for another 90 minutes, the preparation was diluted with 2130 μl of PBS buffer pre-adjusted to pH 8.

This solution was then applied to a PD 10 column ( ^Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer, pH 8, and eluted with PBS buffer, pH 8. The eluate was stirred overnight at room temperature under argon, then concentrated by ultracentrifugation and rediluted with PBS buffer, pH 7.2. Under these conditions, some ADCs may also exist in a closed-ring form. The resulting ADC batch was characterized as follows:

Protein concentration: 1.57 mg/ml

Drug/mAb ratio: 3.7.

Example 234I

Under argon, a solution of 0.029 mg of TCEP in 50 μL of PBS buffer was added to 5 mg of nimotuzumab (c = 10 mg/ml) in 500 μL of PBS. The formulation was stirred at room temperature for 30 minutes, followed by the addition of 0.2 mg (0.00027 mmol) of Intermediate F85 dissolved in 50 μL of DMSO. After stirring at room temperature for an additional 90 minutes, the formulation was diluted with 1900 μL of PBS buffer pre-adjusted to pH 8.

This solution was then applied to a PD 10 column ( ^Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer, pH 8, and eluted with PBS buffer, pH 8. The eluate was stirred overnight at room temperature under argon, then concentrated by ultracentrifugation and rediluted with PBS buffer, pH 7.2. Under these conditions, some ADCs may also exist in a closed-ring form. The resulting ADC batch was characterized as follows:

Protein concentration: 1.93 mg/ml

Drug/mAb ratio: 3.6.

Example 234H

Under argon, a solution of 0.029 mg of TCEP in 50 μL of PBS buffer was added to 5 mg of panitumumab (c = 10 mg/ml) in 500 μL of PBS. The formulation was stirred at room temperature for 4 hours, followed by the addition of 0.2 mg (0.00027 mmol) of intermediate F85 dissolved in 50 μL of DMSO. After stirring at room temperature for an additional 120 minutes, the formulation was diluted with 1900 μL of PBS buffer pre-adjusted to pH 8.

This solution was then applied to a PD 10 column ( ^Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer, pH 8, and eluted with PBS buffer, pH 8. The eluate was stirred overnight at room temperature under argon, then concentrated by ultracentrifugation and rediluted with PBS buffer, pH 7.2. Under these conditions, some ADCs may also exist in a closed-ring form. The resulting ADC batch was characterized as follows:

Protein concentration: 2.12 mg/ml

Drug/mAb ratio: 1.4.

Example 235A

Under argon, a solution of 0.029 mg of TCEP in 50 μl of PBS buffer was added to 5 mg of cetuximab (c = 15.2 mg/ml) in 329 μl of PBS, followed by dilution with 2021 μl of PBS buffer pre-adjusted to pH 8. The formulation was stirred at room temperature for 1 hour, followed by the addition of 0.28 mg of intermediate F235 dissolved in 50 μl of DMSO. After stirring at room temperature for an additional 90 minutes, the formulation was applied to a PD10 column ( ^Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer, pH 8, and eluted with PBS buffer, pH 8. The eluate was stirred at room temperature under argon overnight, then concentrated by ultracentrifugation and rediluted with PBS buffer (pH 7.2). Some of the ADC prepared in this manner may also exist as hydrolyzed open-chain succinamide attached to the antibody. The resulting ADC batch was characterized as follows:

Protein concentration: 1.5 mg/ml

Drug/mAb ratio: 2.4.

Example 235B

Similar to Example 235A, 5 mg of anti-TWEAKR AK-1 (c = 18.6 mg/ml) in 269 μl of PBS was diluted to a concentration of 2 mg/ml using PBS buffer, pH 8, and conjugated to intermediate F235. Some of the ADC prepared in this manner may also exist as hydrolyzed, open-chain succinamide attached to the antibody. The resulting ADC batch was characterized as follows:

Protein concentration: 0.56 mg/ml

Drug/mAb ratio: 1.0.

Example 235E

Similar to Example 235A, 5 mg of trastuzumab (c = 14.5 mg/ml) in 370 μl of PBS was diluted to a concentration of 2 mg/ml using PBS buffer, pH 8, and conjugated to intermediate F235. Some of the ADC prepared in this manner may also exist as hydrolyzed open-chain succinamide attached to the antibody. The resulting ADC batch was characterized as follows:

Protein concentration: 1.67 mg/ml

Drug/mAb ratio: 2.9.

Example 235I

Similar to Example 235A, 5 mg of nimotuzumab (c = 13.08 mg/ml) in 382 μl of PBS was diluted to a concentration of 2 mg/ml using PBS buffer, pH 8, and conjugated to intermediate F235. Some of the ADC prepared in this manner may also exist as hydrolyzed, open-chain succinamide attached to the antibody. The resulting ADC batch was characterized as follows:

Protein concentration: 1.81 mg/ml

Drug/mAb ratio: 2.8.

Example 235H

Similar to Example 235A, 5 mg of panitumumab (c = 67.4 mg/ml) in 74 μl of PBS was diluted to a concentration of 2 mg/ml using PBS buffer, pH 8, and conjugated to intermediate F235. Some of the ADC prepared in this manner may also exist as hydrolyzed, open-chain succinamide attached to the antibody. The resulting ADC batch was characterized as follows:

Protein concentration: 1.14 mg/ml

Drug/mAb ratio: 0.9.

Example 236A

Similar to Example 234A, 5 mg of cetuximab (c = 10 mg/ml) in 500 μl of PBS was coupled to intermediate F236. Under these conditions, some ADC may also exist in a closed-ring form. The resulting ADC batch was characterized as follows:

Protein concentration: 1.85 mg/ml

Drug/mAb ratio: 3.6.

Example 236B

Similar to Example 234A, 5 mg of anti-TWEAKR AK-1 (c = 10 mg/ml) in 500 μl of PBS was coupled to intermediate F236. Under these conditions, some ADC may also exist in a closed-ring form. The resulting ADC batch was characterized as follows:

Protein concentration: 1.67 mg/ml

Drug/mAb ratio: 3.5.

Example 236E

Similar to Example 234A, 5 mg of trastuzumab (c = 10 mg/ml) in 500 μl of PBS was coupled to intermediate F236. Under these conditions, some ADC may also exist in a closed-ring form. The resulting ADC batch was characterized as follows:

Protein concentration: 1.67 mg/ml

Drug/mAb ratio: 3.9.

Example 237A

Under argon, a solution of 0.344 mg of TCEP in 100 μL of PBS buffer was added to 60 mg of cetuximab (c = 15 mg/mL) in 4000 μL of PBS. The formulation was stirred at room temperature for 1 hour, followed by the addition of 3.04 mg (0.003 mmol) of intermediate F209 dissolved in 600 μL of DMSO. After stirring at room temperature for an additional 90 minutes, the formulation was diluted to 15 mL with PBS buffer pre-adjusted to pH 8.

This solution was then applied to a PD 10 column ( ^Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer, pH 8, and eluted with PBS buffer, pH 8. This solution was stirred at room temperature overnight under argon, then concentrated by ultracentrifugation, diluted with PBS buffer (pH 7.2), and reconcentrated. Under these conditions, some ADCs may also exist in a closed-ring form. The resulting ADC batch was characterized as follows:

Protein concentration: 13.78 mg/ml

Drug/mAb ratio: 4.8.

Example 237B

Under argon, 0.287 mg of TCEP in 500 μl of PBS was added to 50 mg of anti-TWEAKR AK-1 (c = 18.6 mg/ml) in 2688 μl of PBS, diluted with 8812 μl of PBS pre-adjusted to pH 8. The formulation was stirred at room temperature for 1 hour, followed by the addition of 2.894 mg (0.003 mmol) of intermediate F209 dissolved in 500 μl of DMSO. After stirring at room temperature for an additional 90 minutes, the formulation was applied to a PD 10 column ( ^Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer, pH 8, and eluted with PBS buffer, pH 8. This solution was stirred at room temperature overnight under argon, then concentrated by ultracentrifugation, diluted with PBS buffer (pH 7.2), and reconcentrated. Under these conditions, some ADC may also exist in the closed-ring form. For this batch, the content of the open-ring form was determined to be 23% directly after synthesis. The resulting ADC batch was characterized as follows:

Protein concentration: 11.1 mg/ml

Drug/mAb ratio: 3.3.

Example 237I

Similar to Example 237A, 60 mg of nimotuzumab (c = 13.08 mg/ml) in 4587 μl of PBS was coupled to intermediate F209. Under these conditions, some ADC may also exist in a closed-ring form. The resulting ADC batch was characterized as follows:

Protein concentration: 15.88 mg/ml

Drug/mAb ratio: 4.0.

Example 238A

Under argon, a solution of 0.029 mg of TCEP in 50 μL of PBS buffer was added to 5 mg of cetuximab (c = 10 mg/ml) in 500 μL of PBS. The formulation was stirred at room temperature for 30 minutes, followed by the addition of 0.22 mg (0.00027 mmol) of intermediate F238 dissolved in 50 μL of DMSO. After stirring at room temperature for an additional 90 minutes, the formulation was diluted with 1900 μL of PBS buffer pre-adjusted to pH 8.

This solution was then applied to a PD 10 column ( ^Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer, pH 8, and eluted with PBS buffer, pH 8. The eluate was stirred at room temperature under argon overnight, then concentrated by ultracentrifugation and rediluted with PBS buffer, pH 7.2. Some of the ADCs prepared in this manner may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody. The resulting ADC batch was characterized as follows:

Protein concentration: 1.89 mg/ml

Drug/mAb ratio: 2.9.

Example 238B

Similar to Example 238A, 5 mg of anti-TWEAKR AK-1 (c = 10 mg/ml) in 500 μl of PBS was conjugated to intermediate F238. Some of the ADC prepared in this manner may also exist as hydrolyzed open-chain succinamide attached to the antibody. The resulting ADC batch was characterized as follows:

Protein concentration: 1.66 mg/ml

Drug/mAb ratio: 3.1.

Under these coupling conditions, 26% of the ADC existed as the hydrolyzed open-chain succinamide attached to the antibody.

Example 238E

Similar to Example 238A, 5 mg of trastuzumab (c = 10 mg/ml) in 500 μl of PBS was conjugated to intermediate F238. Some of the ADC prepared in this manner may also exist as hydrolyzed open-chain succinamide attached to the antibody. The resulting ADC batch was characterized as follows:

Protein concentration: 1.84 mg/ml

Drug/mAb ratio: 3.5.

Example 239A

Under argon, a solution of 0.029 mg of TCEP in 50 μL of PBS buffer was added to 5 mg of cetuximab (c = 10.92 mg/ml) in 458 μL of PBS. The formulation was diluted with 1892 μL of PBS buffer pre-adjusted to pH 8 and stirred at room temperature for 1 hour. Then, 0.19 mg (0.00027 mmol) of intermediate F217 dissolved in 100 μL of DMSO was added. After stirring at room temperature for an additional 90 minutes, the formulation was applied to a PD 10 column ( ^Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer, pH 8, and eluted with PBS buffer, pH 8. The eluate was stirred at room temperature under argon overnight, then concentrated by ultracentrifugation and rediluted with PBS buffer (pH 7.2). Under these conditions, some ADCs may also exist in a closed-ring form. The resulting ADC batch was characterized as follows:

Protein concentration: 1.86 mg/ml

Drug/mAb ratio: 2.8.

Example 239B

Under argon, a solution of 0.029 mg of TCEP in 50 μl of PBS was added to 5 mg of anti-TWEAKR AK-1 (c = 18.6 mg/ml) in 269 μl of PBS. The formulation was diluted with 2081 μl of PBS buffer pre-adjusted to pH 8 and stirred at room temperature for 1 hour. Then, 0.19 mg (0.00027 mmol) of intermediate F217 dissolved in 100 μl of DMSO was added. After stirring at room temperature for an additional 90 minutes, the formulation was applied to a PD 10 column ( ^Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer, pH 8, and eluted with PBS buffer, pH 8. The eluate was stirred at room temperature under argon overnight, then concentrated by ultracentrifugation and rediluted with PBS buffer (pH 7.2). Under these conditions, some ADCs may also exist in a closed-ring form. The resulting ADC batch was characterized as follows:

Protein concentration: 1.28 mg/ml

Drug/mAb ratio: 2.6.

Example 239I

Under argon, a solution of 0.029 mg of TCEP in 50 μL of PBS buffer was added to 5 mg of nimotuzumab (c = 13.1 mg/ml) in 382 μL of PBS. The formulation was diluted with 1968 μL of PBS buffer pre-adjusted to pH 8 and stirred at room temperature for 1 hour. Then, 0.19 mg (0.00027 mmol) of intermediate F217 dissolved in 100 μL of DMSO was added. After stirring at room temperature for an additional 90 minutes, the formulation was applied to a PD 10 column ( ^Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer, pH 8, and eluted with PBS buffer, pH 8. The eluate was stirred at room temperature under argon overnight, then concentrated by ultracentrifugation and rediluted with PBS buffer (pH 7.2). Under these conditions, some ADCs may also exist in a closed-ring form. The resulting ADC batch was characterized as follows:

Protein concentration: 1.32 mg/ml

Drug/mAb ratio: 2.9.

For this ADC preparation, a content of 89% of the open-ring succinamide form was determined.

Example 239H

Under argon, a solution of 0.048 mg of TCEP in 83 μL of PBS buffer was added to 5 mg of panitumumab (c = 67.5 mg/ml) in 74 μL of PBS. The formulation was diluted with 2243 μL of PBS buffer pre-adjusted to pH 8 and stirred at room temperature for 4 hours. 0.19 mg (0.00027 mmol) of intermediate F217 dissolved in 100 μL of DMSO was then added. The formulation was stirred at room temperature overnight. The solution was then applied to a PD 10 column ( ^Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer, pH 8, and eluted with PBS buffer, pH 8. The eluate was stirred at room temperature under argon overnight, then concentrated by ultracentrifugation and rediluted with PBS buffer (pH 7.2). Under these conditions, some ADCs may also exist in a closed-ring form. The resulting ADC batch was characterized as follows:

Protein concentration: 1.33 mg/ml

Drug/mAb ratio: 2.1.

Example 240A

Under argon, a solution of 0.29 mg of TCEP in 500 μL of PBS buffer was added to 50 mg of cetuximab (c = 10.92 mg/ml) in 4579 μL of PBS. The formulation was diluted with 7421 μL of PBS buffer pre-adjusted to pH 8 and stirred at room temperature for 1 hour. Then, 1.4 mg (0.0027 mmol) of intermediate F213 dissolved in 500 μL of DMSO was added. After stirring at room temperature for an additional 90 minutes, the formulation was applied to a PD 10 column ( ^Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer, pH 8, and eluted with PBS buffer, pH 8. The eluate was stirred at room temperature under argon overnight, then concentrated by ultracentrifugation and rediluted with PBS buffer (pH 7.2). Under these conditions, some ADCs may also exist in a closed-ring form. The resulting ADC batch was characterized as follows:

Protein concentration: 15.63 mg/ml

Drug/mAb ratio: 2.7.

Example 240B

Similar to Example 239A, 5 mg of anti-TWEAKR AK-1 (c = 10 mg/ml) in 500 μl of PBS was coupled to intermediate F213. Under these conditions, some ADC may also exist in a closed-ring form. The resulting ADC batch was characterized as follows:

Protein concentration: 0.9 mg/ml

Drug/mAb ratio: 2.0.

Example 240E

Similar to Example 239A, 5 mg of trastuzumab (c = 10 mg/ml) in 500 μl of PBS was coupled to intermediate F213. Under these conditions, some ADC may also exist in a closed-ring form. The resulting ADC batch was characterized as follows:

Protein concentration: 1.4 mg/ml

Drug/mAb ratio: 2.1.

Example 241A

Under argon, a solution of 0.172 mg of TCEP in 300 μL of PBS was added to 30 mg of cetuximab (c = 10 mg/mL) in 3 mL of PBS. The formulation was stirred at room temperature for 30 minutes, followed by the addition of 1.36 mg (1.6 μmol) of intermediate F241 dissolved in 330 μL of DMSO. After stirring at room temperature for 20 hours, the formulation was diluted with 1.37 mL of PBS and eluted using PBS through a PD 10 column ( ^Sephadex® G-25, GE Healthcare). The column was then concentrated by ultracentrifugation, rediluted with PBS (pH 7.2), and reconcentrated. The resulting ADC batch was characterized as follows:

Protein concentration: 9.98 mg/ml

Drug/mAb ratio: 3.3.

Example 241B

Here, 5 mg of anti-TWEAKR AK-1 (c = 10 mg/ml) in PBS was coupled to intermediate F241. After TCEP reduction, the antibody was coupled with stirring overnight and then further processed by Sephadex purification. Following Sephadex purification, the preparation was concentrated by ultracentrifugation and rediluted with PBS.

Protein concentration: 1.39 mg/ml

Drug/mAb ratio: 3.9.

Example 241E

Here, 5 mg of trastuzumab (c = 10 mg/ml) in PBS was conjugated to intermediate F241. After TCEP reduction, conjugation with the antibody was performed with stirring overnight, followed by further workup via Sephadex purification. Following Sephadex purification, the preparation was concentrated by ultracentrifugation and rediluted with PBS.

Protein concentration: 1.83 mg/ml

Drug/mAb ratio: 4.7.

Example 241I

Here, 5 mg of nimotuzumab (c = 10 mg/ml) in PBS was conjugated to intermediate F241. After TCEP reduction, conjugation with the antibody was performed with stirring overnight, followed by further workup via Sephadex purification. Following Sephadex purification, the preparation was concentrated by ultracentrifugation and rediluted with PBS.

Protein concentration: 1.6 mg/ml

Drug/mAb ratio: 4.0.

Example 242A

Under argon, a solution of 0.029 mg of TCEP in 50 μL of PBS buffer was added to 5 mg of cetuximab (c = 10 mg/ml) in 500 μL of PBS. The formulation was stirred at room temperature for 30 minutes, followed by the addition of 0.22 mg (0.00027 mmol) of intermediate F242 dissolved in 50 μL of DMSO. After stirring at room temperature for an additional 90 minutes, the formulation was diluted with 1900 μL of PBS buffer pre-adjusted to pH 8.

This solution was then applied to a PD 10 column ( ^Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer, pH 8, and eluted with PBS buffer, pH 8. The eluate was stirred overnight at room temperature under argon, then concentrated by ultracentrifugation and rediluted with PBS buffer, pH 7.2. Under these conditions, some ADCs may also exist in a closed-ring form. The resulting ADC batch was characterized as follows:

Protein concentration: 1.92 mg/ml

Drug/mAb ratio: 2.7.

Example 242B

As described in Example 242A, 5 mg of anti-TWEAKR AK-1 (c = 10 mg/ml) in 500 μl of PBS was coupled to 0.22 mg of intermediate F242. Under these conditions, some ADC may also exist in a closed-ring form. The resulting ADC batch was characterized as follows:

Protein concentration: 1.54 mg/ml

Drug/mAb ratio: 3.1.

Example 242E

As described in Example 242A, 5 mg of trastuzumab (c = 10 mg/ml) in 500 μl of PBS was coupled to 0.22 mg of intermediate F242. Under these conditions, some ADC may also exist in a closed-ring form. The resulting ADC batch was characterized as follows:

Protein concentration: 1.76 mg/ml

Drug/mAb ratio: 3.6.

Example 243A

Under argon, a solution of 0.029 mg of TCEP in 50 μL of PBS buffer was added to 5 mg of cetuximab (c = 10 mg/ml) in 500 μL of PBS. The formulation was stirred at room temperature for 30 minutes, followed by the addition of 0.23 mg (0.00027 mmol) of intermediate F243 dissolved in 50 μL of DMSO. After stirring at room temperature for an additional 90 minutes, the formulation was diluted with 1900 μL of PBS buffer pre-adjusted to pH 8.

This solution was then applied to a PD 10 column ( ^Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer, pH 8, and eluted with PBS buffer, pH 8. The eluate was stirred overnight at room temperature under argon, then concentrated by ultracentrifugation and rediluted with PBS buffer, pH 7.2. Under these conditions, some ADCs may also exist in a closed-ring form. The resulting ADC batch was characterized as follows:

Protein concentration: 1.92 mg/ml

Drug/mAb ratio: 2.9.

Example 243B

As described in Example 243A, 5 mg of anti-TWEAKR AK-1 (c = 10 mg/ml) in 500 μl of PBS was coupled to 0.23 mg of intermediate F243. Under these conditions, some ADC may also exist in a closed-ring form. The resulting ADC batch was characterized as follows:

Protein concentration: 1.63 mg/ml

Drug/mAb ratio: 3.1.

Example 243E

As described in Example 243A, 5 mg of trastuzumab (c = 10 mg/ml) in 500 μl of PBS was coupled to 0.23 mg of intermediate F243. Under these conditions, some ADC may also exist in a closed-ring form. The resulting ADC batch was characterized as follows:

Protein concentration: 1.8 mg/ml

Drug/mAb ratio: 3.5.

Example 243I

As described in Example 243A, 5 mg of nimotuzumab (c = 10 mg/ml) in 500 μl of PBS was coupled to 0.23 mg of intermediate F243. Under these conditions, some ADC may also exist in a closed-ring form. The resulting ADC batch was characterized as follows:

Protein concentration: 1.87 mg/ml

Drug/mAb ratio: 3.1.

Example 244A

Here, 5.0 mg of cetuximab (c = 23.10 mg/ml) in PBS was used for coupling to intermediate F244, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted.

Protein concentration: 2.12 mg/ml

Drug/mAb ratio: 3.5.

Example 244B

Here, 5.0 mg of anti-TWEAK AK-1 (c = 18.60 mg/ml) in PBS was used for coupling to intermediate F244, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted.

Protein concentration: 1.65 mg/ml

Drug/mAb ratio: 3.5.

Example 244E

Here, 5.0 mg of trastuzumab (c = 13.50 mg/ml) in PBS was used for coupling to intermediate F244, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted.

Protein concentration: 1.91 mg/ml

Drug/mAb ratio: 3.5.

Example 244I

Here, 5.0 mg of nimotuzumab (c = 13.08 mg/ml) in PBS was used for coupling to intermediate F244, and the formulation was concentrated by ultracentrifugation and rediluted after Sephadex purification.

Protein concentration: 1.91 mg/ml

Drug/mAb ratio: 2.9.

Example 245A

Under argon, a solution of 0.029 mg of TCEP in 50 μL of PBS buffer was added to 5 mg of cetuximab (c = 10 mg/ml) in 500 μL of PBS. The formulation was stirred at room temperature for 30 minutes, followed by the addition of 0.24 mg (0.00027 mmol) of intermediate F245 dissolved in 50 μL of DMSO. After stirring at room temperature for an additional 90 minutes, the formulation was diluted with 1900 μL of PBS buffer pre-adjusted to pH 8.

This solution was then applied to a PD 10 column ( ^Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer, pH 8, and eluted with PBS buffer, pH 8. The eluate was stirred overnight at room temperature under argon, then concentrated by ultracentrifugation and rediluted with PBS buffer, pH 7.2. Under these conditions, some ADCs may also exist in a closed-ring form. The resulting ADC batch was characterized as follows:

Protein concentration: 1.69 mg/ml

Drug/mAb ratio: 2.4.

Example 245B

As described in Example 245A, 5 mg of anti-TWEAKR AK-1 (c = 10 mg/ml) in 500 μl of PBS was coupled to 0.24 mg of intermediate F245. Under these conditions, some ADC may also exist in a closed-ring form. The resulting ADC batch was characterized as follows:

Protein concentration: 1.51 mg/ml

Drug/mAb ratio: 2.5.

Example 245E

As described in Example 245A, 5 mg of trastuzumab (c = 10 mg/ml) in 500 μl of PBS was coupled to 0.24 mg of intermediate F245. Under these conditions, some ADC may also exist in a closed-ring form. The resulting ADC batch was characterized as follows:

Protein concentration: 1.8 mg/ml

Drug/mAb ratio: 3.5.

Example 245I

As described in Example 245A, 5 mg of nimotuzumab (c = 10 mg/ml) in 500 μl of PBS was coupled to 0.24 mg of intermediate F245. Under these conditions, some ADC may also exist in a closed-ring form. The resulting ADC batch was characterized as follows:

Protein concentration: 1.65 mg/ml

Drug/mAb ratio: 2.3.

Example 246

4-[(2-{[(2R)-2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butanoyl}amino)-2-carboxyethyl]amino}-2-oxoethyl)amino]-3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-4-oxobutanoic acid/trifluoroacetic acid (1:1)

and

4-[(2-{[(2R)-2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butanoyl}amino)-2-carboxyethyl]amino}-2-oxoethyl)amino]-2-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-4-oxobutanoic acid/trifluoroacetic acid (1:1)

First, L-cysteine is converted to N-{[ 2-(trimethylsilyl)ethoxy]carbonyl}-L-cysteine using 1-({[2-(trimethylsilyl)ethoxy]carbonyl}oxy)pyrrolidine-2,5-dione in the presence of N,N-diisopropylethylamine in DMF.

11 mg (0.013 mmol) of intermediate F193 and 8 mg (0.016 mmol) of N-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-cysteine were dissolved in 3 ml of DMF and the mixture was stirred at room temperature for 20 h. The mixture was then concentrated and the residue was purified by preparative HPLC.

After combining the appropriate fractions and evaporating the solvent under vacuum, the residue was dissolved in 2 ml of THF/water (1:1). 19 μl of 2M aqueous lithium hydroxide solution was added, and the preparation was stirred at room temperature for 1 hour. An additional 19 μl of 2M aqueous lithium hydroxide solution was then added, and the preparation was stirred at room temperature overnight. The mixture was then neutralized with 1M hydrochloric acid, the solvent evaporated under vacuum, and the residue purified by preparative HPLC. This yielded 4.1 mg (38% of theory) of the regioisomeric protected intermediate as a colorless foam.

LC-MS (Method 1): R _t = 1.03 min (broad); MS (ESIpos): m/z = 1020 (M+H) ⁺ .

In the final step, 4.1 mg (0.004 mmol) of this intermediate was dissolved in 3 ml of 2,2,2-trifluoroethanol. 3 ml (0.022 mmol) of zinc chloride was added, and the preparation was stirred at 50°C for 1 hour. Then, 6 mg (0.022 mmol) of ethylenediamine-N,N,N',N'-tetraacetic acid and 2 ml of 0.1% aqueous trifluoroacetic acid were added, and the solvent was evaporated under vacuum. The residue was purified by preparative HPLC. Concentration of the appropriate fractions and lyophilization of the residue from acetonitrile/water yielded 5 mg (quantitative) of the title compound as a 20:80 mixture of regioisomers.

LC-MS (Method 1): R _t = 0.78 min (broad); MS (ESIpos): m/z = 876 (M+H) ⁺ .

LC-MS (Method 5): R _t = 2.36 min and 2.39 min; MS (ESIpos): m/z = 876 (M+H) ⁺ .

Example 247A

Under argon, a solution of 0.029 mg of TCEP in 50 μL of PBS was added to 5 mg of cetuximab (c = 10 mg/mL) in 500 μL of PBS. The formulation was stirred at room temperature for 30 minutes, followed by the addition of 0.264 mg (0.27 μmol) of intermediate F247 dissolved in 50 μL of DMSO. After stirring at room temperature for 20 hours, the formulation was diluted with 1.9 mL of PBS and eluted using PBS through a PD 10 column ( ^Sephadex® G-25, GE Healthcare). The column was then concentrated by ultracentrifugation, rediluted with PBS (pH 7.2), and reconcentrated. The resulting ADC batch was characterized as follows:

Protein concentration: 1.66 mg/ml

Drug/mAb ratio: 2.2.

Example 247B

Here, 5 mg of anti-TWEAKR AK-1 (c = 10 mg/ml) in PBS was used for coupling to intermediate F247, and coupling and workup were performed as described in Example 247A.

Protein concentration: 1.49 mg/ml

Drug/mAb ratio: 2.6.

Example 247E

Here, 5 mg of trastuzumab (c = 10 mg/ml) in PBS was used for coupling to intermediate F247, and coupling and workup were performed as described in Example 247A.

Protein concentration: 1.67 mg/ml

Drug/mAb ratio: 2.3.

Example 247I

Here, 5 mg of nimotuzumab (c = 10 mg/ml) in PBS was used for coupling to intermediate F247, and coupling and workup were performed as described in Example 247A.

Protein concentration: 1.62 mg/ml

Drug/mAb ratio: 2.4.

Example 248A

Here, similar to Example 5A, 5 mg of cetuximab (c = 10.92 mg/ml) in PBS was used for coupling with intermediate F248, and the formulation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some of the ADC prepared in this way may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 2.06 mg/ml

Drug/mAb ratio: 3.8.

Example 248B

Here, similar to Example 5B, 5 mg of anti-TWEAKR AK-1 (c = 18.6 mg/ml) in PBS was used for coupling with intermediate F248, and the preparation was concentrated by ultracentrifugation after Sephadex purification and rediluted with PBS. Some of the ADC prepared in this way may also exist in the form of hydrolyzed open-chain succinamide attached to the antibody.

Protein concentration: 1.84 mg/ml

Drug/mAb ratio: 4.1.

Example 249

S-{1-[6-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)hexyl]-2,5-dioxopyrrolidin-3-yl}-L-cysteine/trifluoroacetic acid (1:1)

11 mg (14 μmol) of intermediate F179 were placed in 2.2 ml of DMF and 3.3 mg (27 μmol) of L-cysteine were added. The reaction mixture was stirred at room temperature for 3 hours and then concentrated under vacuum. The residue was purified by preparative HPLC. Concentration of the appropriate fractions and lyophilization of the residue from acetonitrile/water gave 7.3 mg (58% of theoretical) of the title compound as a colorless foam.

LC-MS (Method 4): R _t = 1.04 min; MS (EIpos): m/z = 813 [M+H] ⁺ .

Example 250

4-{[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butanoyl}amino)ethyl]amino}-3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-4-oxobutanoic acid/trifluoroacetic acid (1:1)

and

4-{[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butanoyl}amino)ethyl]amino}-2-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-4-oxobutanoic acid/trifluoroacetic acid (1:1)

10 mg (0.013 mmol) of intermediate F85 and 5.3 mg (0.02 mmol) of N-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-cysteine were dissolved in 3 ml of DMF and stirred at room temperature for 3 days. The mixture was then concentrated, and the residue was purified by preparative HPLC. After combining the appropriate fractions and evaporating the solvent under vacuum, the residue was dissolved in 2 ml of THF/water (1:1). 9 μl of 2M aqueous lithium hydroxide solution was added, and the mixture was stirred at room temperature for 1 hour. The mixture was then adjusted to pH ~3 with 1M hydrochloric acid, the solvent evaporated under vacuum, and the residue purified by preparative HPLC. This yielded 3 mg (24% of theory over 2 steps) of the regioisomeric protected intermediate as a colorless foam.

LC-MS (Method 5): R _t = 3.39 min and 3.43 min; MS (ESIpos): m/z = 919 (M+H) ⁺ .

In the final step, 3 mg (0.0033 mmol) of this intermediate was dissolved in 3 ml of 2,2,2-trifluoroethanol. 2.2 ml (0.016 mmol) of zinc chloride was added, and the preparation was stirred at 50°C for 3.5 hours. Then, 4.8 mg (0.016 mmol) of ethylenediamine-N,N,N',N'-tetraacetic acid was added, and the solvent was evaporated under vacuum. The residue was purified by preparative HPLC. Concentration of the appropriate fractions and lyophilization of the residue from acetonitrile/water yielded 1 mg (33% of theory) of the title compound as a 43:34 regioisomer mixture. This isomer mixture contained 23% of the other isomer (RT = 2.51).

LC-MS (Method 5): R _t = 2.57 min and 2.62 min; MS (ESIpos): m/z = 775 (M+H) ⁺ .

Example 251

S-(1-{2-[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethoxy]ethyl}-2,5-dioxopyrrolidin-3-yl)-L-cysteine/trifluoroacetic acid (1:1)

3 mg (4 μmol) of intermediate F248 were placed in 2 ml of DMF and 0.9 mg (8 μmol) of L-cysteine was added. The reaction mixture was stirred at room temperature for 18 hours and then concentrated under vacuum. The residue was purified by preparative HPLC. Concentration of the appropriate fractions and lyophilization of the residue from acetonitrile/water gave 1.1 mg (32% of theory) of the title compound as a white solid.

LC-MS (Method 1): R _t = 0.78 min; MS (EIpos): m/z = 801 [M+H] ⁺ .

Example 252

(3R,7S)-7-Amino-17-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-3-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-4-glycoloyl-2,2-dimethyl-8,16-dioxo-12-oxa-4,9,15-triazanonadecan-19-oic acid/trifluoroacetic acid (1:1)

and

(3R,7S)-7-Amino-18-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-3-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-4-glycoloyl-2,2-dimethyl-8,16-dioxo-12-oxa-4,9,15-triazanonadecan-19-oic acid/trifluoroacetic acid (1:1)

8 mg (0.010 mmol) of the protected intermediate of Intermediate F248 and 5.1 mg (0.02 mmol) of N-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-cysteine were dissolved in 3 mL of DMF and stirred at room temperature for 18 hours, followed by treatment in an ultrasonic bath for 2 hours. The mixture was then concentrated, and the residue was purified by preparative HPLC. After combining the appropriate fractions and evaporating the solvent under vacuum, the residue was dissolved in 2 mL of THF/water (1:1). 15 μL of 2M aqueous lithium hydroxide solution was added, and the mixture was stirred at room temperature for 15 minutes. The mixture was then adjusted to pH ~3 with 1M hydrochloric acid, diluted with 20 mL of sodium chloride solution, and extracted twice with 20 mL of ethyl acetate. The organic phase was dried over magnesium sulfate and concentrated, and the residue was lyophilized from acetonitrile/water. This yielded 8.4 mg (78% of theory over 2 steps) of the regioisomeric protected intermediate as a colorless foam.

LC-MS (method 1): R _t = 1.44 min and 3.43 min; MS (ESIpos): m/z = 1107 (M+H) ⁺ .

In the final step, 8 mg (0.007 mmol) of this intermediate was dissolved in 5 ml of 2,2,2-trifluoroethanol. 9.8 ml (0.072 mmol) of zinc chloride was added, and the preparation was stirred at 50°C for 1.5 hours. Ethylenediamine-N,N,N',N'-tetraacetic acid was then added, and the solvent was evaporated under vacuum. The residue was purified by preparative HPLC. Concentration of the appropriate fractions and lyophilization of the residue from acetonitrile/water yielded 4 mg (59% of theory) of the title compound as a 31:67 mixture of regioisomers.

LC-MS (method 1): R _t = 0.79 min and 0.81 min; MS (ESIpos): m/z = 819 (M+H) ⁺ .

Example 253

4-({2-[(N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl)amino]ethyl}amino)-3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-4-oxobutanoic acid/trifluoroacetic acid (1:1)

and

4-({2-[(N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butyryl}-β-alanyl)amino]ethyl}amino)-2-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-4-oxobutanoic acid/trifluoroacetic acid (1:1)

The isomeric title compounds were prepared in analogy to Example 250 from intermediate F84 and N-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-cysteine.

Example 254A

Under argon, a solution of 0.029 mg of TCEP in 50 μL of PBS was added to 5 mg of cetuximab (c = 10 mg/mL) in 500 μL of PBS. The formulation was stirred at room temperature for 30 minutes, followed by the addition of 0.264 mg (0.27 μmol) of intermediate F254 dissolved in 50 μL of DMSO. After stirring at room temperature for 20 hours, the formulation was diluted with 1.9 mL of PBS and eluted using PBS through a PD 10 column ( ^Sephadex® G-25, GE Healthcare). The column was then concentrated by ultracentrifugation, rediluted with PBS (pH 7.2), and reconcentrated. The resulting ADC batch was characterized as follows:

Protein concentration: 1.74 mg/ml

Drug/mAb ratio: 2.2.

Example 254B

Here, 5 mg of anti-TWEAKR AK-1 (c = 10 mg/ml) in PBS was used for coupling to intermediate F254, and coupling and workup were performed as described in Example 254A.

Protein concentration: 1.8 mg/ml

Drug/mAb ratio: 2.5.

Example 254E

Here, 5 mg of trastuzumab (c = 10 mg/ml) in PBS was used for coupling to intermediate F254, and coupling and workup were performed as described in Example 254A.

Protein concentration: 1.74 mg/ml

Drug/mAb ratio: 2.4.

Example 254I

Here, 5 mg of nimotuzumab (c = 10 mg/ml) in PBS was used for coupling to intermediate F254, and coupling and workup were performed as described in Example 254A.

Protein concentration: 1.73 mg/ml

Drug/mAb ratio: 2.0.

Example 255

(2R,28R)-28-amino-2-[({2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}sulfanyl)methyl]-25-(carboxymethyl)-4,20,24-trioxo-7,10,13,16-tetraoxa-26-thia-3,19,23-triazanonacosane-1,29-dioic acid/trifluoroacetic acid (1:2) and

(1R,28R,34R)-1-amino-33-(3-aminopropyl)-34-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-35,35-dimethyl-6,10,26,32-trioxo-14,17,20,23-tetraoxa-3,30-dithia-7,11,27,33-tetraazahexatriacontane-1,4,28-tricarboxylic acid/trifluoroacetic acid (1:2)

20 mg (0.018 mmol) of R-{2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}-N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecanadecanoyl]-L-cysteine/trifluoroacetic acid (1:1) (Intermediate F209) and 9.78 mg (0.036 mmol) of N-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-cysteine were dissolved in 2 ml of DMF and the mixture was stirred at room temperature for 18 hours. The reaction mixture was evaporated under vacuum. The residue (47.7 mg) was dissolved in 3 mL of THF/water (1:1). 0.08 mL of 2M aqueous lithium hydroxide solution was added, and the mixture was stirred at room temperature for 1 hour. The pH of the mixture was then adjusted to ~7 using 9.26 mg (0.15 mmol) of acetic acid. The reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 125x30; 10µ, flow rate: 50 mL/min, MeCN/water; 0.1% TFA). The solvent was evaporated under vacuum, and the residue was dried under high vacuum. This yielded 15.3 mg (29% over 2 steps) of the regioisomeric protected intermediate.

LC-MS (Method 6): R _t = 12.26 min and 12.30 min; MS (ESIpos): m/z = 1254 (M+H) ⁺ .

In the final step, 15.3 mg (0.01 mmol) of this intermediate was dissolved in 2 ml of 2,2,2-trifluoroethanol. 6.1 ml (0.05 mmol) of zinc chloride was added, and the preparation was stirred at 50°C for 2 hours. Then, 13.1 mg (0.05 mmol) of ethylenediamine-N,N,N',N'-tetraacetic acid was added, and the solvent was purified by preparative HPLC. Concentration of the appropriate fractions and lyophilization of the residue from acetonitrile/water yielded 11.9 mg (79.5%) of the title compound as a mixture of regioisomers.

LC-MS (Method 1): R _t = 0.85 min; MS (ESIpos): m/z = 1110 (M+H) ⁺ .

Example 256

(3R)-6-{(11S,15R)-11-amino-15-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-14-glycoloyl-16,16-dimethyl-2,5,10-trioxo-3,6,9,14-tetraazaheptadecan-1-yl}-5-oxothiomorpholine-3-carboxylic acid/trifluoroacetic acid (1:1)

4 mg (0.004 mmol) of the compound from Example 135 were dissolved in 4 ml of THF/water and 48 microliters of 2 molar lithium hydroxide solution were added. The preparation was stirred at room temperature for 1 hour, then concentrated and purified by preparative HPLC. Combining the appropriate fractions, concentrating them and lyophilizing them from acetonitrile/water gave 2.4 mg (60% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.86 min; MS (EIpos): m/z = 814 [M+H] ⁺ .

C: Evaluation of biological efficacy

The biological effects of the compounds of the invention can be demonstrated in the following assays:

m. Determination of the cytotoxicity of C-1a anti-TWEAKR ADC

Analysis of the cytotoxic effect of anti-TWEAKR-ADC using various cell lines:

NCI-H292: human mucoepidermoid lung cancer cells, ATCC-CRL-1848, cultured in standard medium: RPMI 1640 (Biochrom; #FG1215, stabilized (stab.) glutamine) + 10% FCS (Biochrom; #S0415), TWEAKR positive, EGFR positive.

BxPC3: human pancreatic cancer cells, ATCC-CRL-1687, cultured in RPMI 1640 (Biochrom; #FG1215, stabilized glutamine) + 10% FCS (Biochrom; #S0415), TWEAKR positive.

KPL4: human breast cancer cells, standard culture medium: RPMI 1640 + GlutaMAX I + 10% FBS, cell bank, Bayer Pharma AG (verified and confirmed at DSMZ on July 19, 2012), Berlin, ERBB2 positive.

Cells were cultured by standard methods as indicated by the American Tissue Type Collection (ATCC) for the respective cell lines.

MTT assay

The assay is performed by detaching cells with Accutase in PBS (Biochrom AG #L2143), pelleting them, resuspending them in culture medium, counting them, and seeding them into white-bottomed 96-well plates (Costar #3610) (1000–2000 cells, 100 µl/well, depending on the cell type). The cells are then incubated at 37°C and 5% CO2 in an incubator. After 48 hours, the antibody-active substance conjugate is added to the cells in triplicate at concentrations ranging from 10 ^{^-5} M to 10^{^-13} M in 10 µl of culture medium and incubated at 37°C and 5% CO2. After 72 hours, proliferation is measured using the MTT assay (ATCC, Manassas, Virginia, USA, Catalog No. 30-1010K). Following the selected incubation period, the cells are incubated with the MTT reagent for 4 hours, followed by overnight lysis by the addition of detergent. The resulting dye is detected at 570 nm. The proliferation of cells not treated with the test substances but treated identically in other respects was defined as a value of 100%.

CTG test

Cells were cultured according to standard protocols using the culture medium listed under C-1. The assay was performed by detaching cells using a solution of trypsin (0.05%) and EDTA (0.02%) in PBS (Biochrom AG #L2143), pelleting, resuspending in culture medium, counting, and seeding into white-bottomed 96-well plates (Costar #3610) (75 µl/well, with the following cell numbers per well: NCI-H292: 2500 cells/well, BxPC3: 2500 cells/well). The cells were then incubated in a 37°C incubator under 5% carbon dioxide. After 24 hours, the antibody-active substance conjugate was added to the cells in 25 µl of culture medium (fourfold concentrated) to provide a final antibody-active substance conjugate concentration of 3 x ^10⁻⁷ to 3 x ^10⁻¹⁰ M on the cells (in triplicate). The cells were then incubated in a 37°C incubator under 5% carbon dioxide. Cell viability at the start of active substance treatment (day 0) was determined in parallel plates using the Cell Titer Glow (CTG) Luminescent Cell Viability Assay (Promega #G7573 and #G7571). To this end, 100 μL of substrate was added per cell batch, and the plates were then covered with aluminum foil and shaken on a plate shaker at 180 rpm for 2 minutes. The plates were then allowed to rest on the bench for 8 minutes before being measured using a luminometer (Victor X2, Perkin Elmer). The substrate detects the ATP content of viable cells, generating a luminescent signal whose intensity is proportional to cell viability. After 72 hours of incubation with the antibody-active substance conjugates, cell viability was also determined using the Cell Titer Glow Luminescent Cell Viability Assay as described above. The measured data were used to calculate the _IC50 for growth inhibition compared to day 0 using a DRC (dose-response curve) analysis spreadsheet and a 4-parameter fit. The DRC analysis spreadsheet is a Biobook spreadsheet developed by Bayer Pharma AG and Bayer Business Services on the IDBS E-WorkBook Suite platform (IDBS: ID Business Solutions Ltd., Guildford, UK).

Tables 1a, 1b, and 1c below list _IC50 values for representative examples from this assay using anti-TWEAKR antibodies:

.

The activity data shown relate to the examples described in this experimental section and indicate the drug/mAb ratio. These values may be deviated at different drug/mAb ratios. IC50 values are averages or individual values from several independent experiments. The activity of the TWEAKR antibody/active substance conjugates is selective for the respective isotype controls comprising the respective linker and toxin cluster.

Table 2 below lists the IC50 values of representative examples using cetuximab antibodies from the MTT assay:

.

The action data shown relate to the examples described in this experimental section and are based on the drug/mAb ratios. These values may be deviated at different drug/mAb ratios. IC50 values are averages or individual values from several independent experiments. The effects of the cetuximab antibody/active substance conjugates were selective for their respective isotype controls containing their respective linkers and toxin clusters.

Table 3 below lists the IC50 values from the MTT assay for representative examples using the trastuzumab antibody:

.

The effect data shown relate to the examples described in this experimental section and are based on the drug/mAb ratios. These values are optionally offset at different drug/mAb ratios. IC50 values are averages or individual values from several independent experiments. The effect of the trastuzumab antibody/active substance conjugates was selective for their respective isotype controls containing their respective linkers and toxin clusters.

C-1b Determination of the Inhibition of Spindle Kinesin KSP/Eg5 by Selected Examples

The motor domain of human spindle kinesin KSP/Eg5 (tebu-bio/Cytoskeleton Inc., No. 027EG01-XL) was incubated at a concentration of 10 nM in 15 mM PIPES, pH 6.8 (5 mM _MgCl₂ and 10 mM DTT, Sigma) with tubulin (bovine or porcine, tebu-bio/Cytoskeleton Inc.) stabilized with 50 µg/ml paclitaxel (Sigma No. T7191-5MG) at room temperature for 5 minutes. The freshly prepared mixture was aliquoted into 384-well MTP plates. The inhibitor of interest was then added at concentrations ranging from 1.0 x ^10⁻⁶ M to 1.0 x ^10⁻⁶ M and ATP (final concentration 500 µM, Sigma). The cells were incubated at room temperature for 2 hours. ATPase activity was determined by measuring the formation of inorganic phosphate using malachite green (Biomol). After adding the reagent, the sample was incubated at room temperature for 50 minutes before measuring absorbance at 620 nm. Monastrol and ispinesib (Adooq A10486) were used as positive controls. Data for each dose-effect curve were determined eightfold. IC50 values are the mean of three independent experiments. The 100% control was a sample untreated with the inhibitor.

Table 4 below summarizes the IC50 values of representative examples from the assay; they demonstrate in an exemplary manner the high potency of the toxic cluster and ADC metabolites at the target.

The performance data shown relate to the examples described in this experimental part.

C-2 internalization assay

Internalization is a key process that enables specific and efficient delivery of cytotoxic payloads to antigen-expressing cancer cells via antibody-drug conjugates (ADCs). This process was monitored by fluorescent labeling with a specific TWEAKR antibody and an isotype control antibody (M014). First, a fluorescent dye was conjugated to a lysine residue on the antibody. Conjugation was performed using a two-fold molar excess of CypHer5E monoNHS ester (Batch 357392, GE Healthcare) at pH 8.3. Following conjugation, the reaction mixture was dialyzed overnight at 4°C (sSlide-A-Lyser Dialysis Cassettes MWCD 10kD, Pierce) to remove excess dye and adjust the pH. The protein solution was then concentrated (VIVASPIN 500, Sartorius stedimbiotec). Antibody dye loading was determined by spectrophotometric analysis (NanoDrop) and subsequent calculation (D:P = A _dye ε _protein : (A ₂₈₀ - 0.16 _{A dye} ) ε _dye ). Dye loading was comparable between the TWEAKR antibodies and isotype controls examined here, and in cell-binding assays, it was demonstrated that the conjugation did not alter antibody affinity.

This labeled antibody was used for internalization detection. Prior to treatment, cells (2 x ^10⁴ /well) were seeded in 100 μl of culture medium in a 96-well MTP (thick, black, clear bottom, No. 4308776, Applied Biosystems). After 18 hours of incubation at 37°C/5% _CO₂ , the medium was replaced and labeled anti-TWEAKR antibody was added at various concentrations (10, 5, 2.5, 1, and 0.1 μg/ml). The same treatment procedure was followed for a labeled isotype control (negative control). Incubation times were 0, 0.25, 0.5, 1, 1.5, 2, 3, 6, and 24 hours. Fluorescence was measured using an InCellanalyser 1000 (GE Healthcare). Kinetics were then assessed using the parameters particle count/cell and total particle intensity/cell.

After binding to TWEAKR, the internalization capacity of TWEAKR antibodies was examined. To this end, cells with different TWEAKR expression levels were selected. Target-mediated specific internalization was observed with TWEAKR antibodies, while isotype controls showed no internalization.

C-3 In vitro assay for measuring cell permeability

The cell permeability of substances can be investigated in vitro using flux assays with Caco-2 cells [MD Troutman and DR Thakker, Pharm. Res. 20 (8) , 1210-1224 (2003)]. To this end, cells are cultured for 15-16 days on 24-well filter plates. To determine the permeation, the test substance is applied to the cells apically (A) or basally (B) in HEPES buffer and incubated for 2 hours. After 0 hours and after 2 hours, samples are taken from the cis and trans compartments. The samples are separated by HPLC using a reversed-phase column (Agilent 1200, Böblingen, Germany). The HPLC system is coupled to a triple quadrupole mass spectrometer API 4000 (Applied Biosystems Applera, Darmstadt, Germany) via a Turbo IonSpray interface. Permeability was assessed based on P _app values calculated using the formula published by Schwab et al. [D. Schwab et al., J. Med. Chem. 46, 1716-1725 (2003)]. Substances were classified as actively transported when the ratio of P _app (BA) to P _app (AB) (efflux rate) was >2 or <0.5.

Crucial for intracellularly released toxic clusters are the permeability from B to A [P _app (BA)] and the ratio of P _app (BA) to P _app (AB) (efflux rate): The lower this permeability, the slower the active and passive transport of the substance across the Caco-2 cell monolayer. If the efflux rate does not otherwise indicate any active transport, the substance remains in the cell longer after intracellular release. Therefore, it has more time to interact with its biochemical target (in this case, the spindle kinesin, KSP/Eg5).

Table 5 below lists permeability data from representative examples of this assay:

.

C-4 In vitro assay for determining the substrate properties of P-glycoprotein (P-gp)

Many tumor cells express transporters for active substances, which often leads to the development of resistance to cytostatics. Substances that are not substrates for such transporters, such as P-glycoprotein (P-gp) or BCRP, can therefore exhibit an improved profile of action.

The substrate properties of substances for P-gp (ABCB1) were determined using a flux assay using LLC-PK1 cells (L-MDR1 cells) overexpressing P-gp [AH Schinkel et al., J. Clin. Invest. 96, 1698-1705 (1995)]. LLC-PK1 cells or L-MDR1 cells were cultured in 96-well filter plates for 3-4 days. To determine permeation, the test substances were applied to the cells apically (A) or basally (B) in HEPES buffer, either alone or in the presence of an inhibitor (e.g., ivermectin or verapamil), and incubated for 2 hours. Samples were taken from the cis and trans compartments after 0 and 2 hours. The samples were separated by HPLC using a reversed-phase column. The HPLC system was coupled to a triple quadrupole mass spectrometer API 3000 (Applied Biosystems Applera, Darmstadt, Germany) via a Turbo Ion Spray interface. Permeability was assessed based on P _app values calculated using the formula published by Schwab et al. [D. Schwab et al., J. Med. Chem. 46, 1716-1725 (2003)]. Substances were classified as P-gp substrates when the efflux ratio of P _app (BA) to P _app (AB) was >2.

As a further criterion for assessing the nature of a P-gp substrate, the efflux rates in L-MDR1 and LLC-PK1 cells or in the presence or absence of an inhibitor can be compared. If these values differ by more than a factor of 2, the substance in question is a P-gp substrate.

C-5 pharmacokinetics

C5a: Identification of ADC metabolites after in vitro internalization

Description of the method:

Internalization studies using immunoconjugates were performed to analyze intracellular metabolite formation. To this end, human lung tumor NCI H292 cells ( ^3x10⁵ /well) were seeded in 6-well plates and cultured overnight (37°C, 5% _CO₂ ). The cells were treated with 10 µg/mL of the ADC to be tested. Internalization was performed at 37°C and 5% _CO₂ . At various time points (0, 4, 24, 48, and 72 hours), cell samples were collected for further analysis. First, the supernatant (approximately 5 mL) was collected and centrifuged (2 minutes at room temperature, 1000 rpm using a Heraeus Variofuge 3.0R) before being stored at -80°C. The cells were washed with PBS, detached with Accutase, and the cell number was determined. After another wash, 100 μL of lysis buffer (Mammalian Cell Lysis Kit (Sigma MCL1)) was added to the indicated number of cells (2 x 10 ⁵ ) and incubated in Protein LoBind tubes (Eppendorf Catalog No. 0030 108.116) with continuous shaking (Homogenizer, 15 min, 4°C, 650 rpm). Following incubation, the lysate was centrifuged (10 min, 4°C, 12,000 g, Eppendorf 5415R) and the supernatant was collected. The resulting supernatant was stored at –80°C. All samples were then analyzed as follows.

Compounds were measured in culture supernatants or cell lysates by high-pressure liquid chromatography (HPLC) coupled to triple quadrupole mass spectrometry (MS) after protein precipitation with methanol or acetonitrile.

For post-processing of 50 μL of culture supernatant/cell lysate, add 150 μL of precipitation reagent (usually acetonitrile) and vortex for 10 seconds. The precipitation reagent should contain an internal standard (ISTD) at an appropriate concentration (usually 20-100 ng/mL). After centrifugation at 16,000 g for 3 minutes, transfer the supernatant to an autosampler vial, add 500 μL of the appropriate buffer for the eluent, and vortex again.

Finally both matrix samples were measured using HPLC coupled to a triple quadrupole mass spectrometer API 6500 from AB SCIEX Deutschland GmbH.

For calibration, plasma samples were spiked with concentrations ranging from 0.5 to 2000 µg/l. The limit of detection (LOQ) was approximately 2 µg/l. The linear range extended from 2 to 1000 µg/l.

To calibrate the tumor samples, concentrations of 0.5–200 µg/l were spiked into untreated tumor supernatant. The limit of detection was 4 µg/l. The linear range extended from 4 to 200 µg/l.

The quality controls (Qualitätskontrollen) used to test the effectiveness contained 5 and 50 µg/l.

NCI-H292 cells were incubated with 10 µg/ml of each of the ADCs from Examples 104b, 119b, 155b, 165b, and 173b. After 72 hours, the cells were washed with PBS, lysed, and deep-frozen (-80°C). Using the methods described above, cell lysates and cell culture supernatants were post-processed and the following metabolites were identified and quantified after extraction:

.

NCI-H292 cells were incubated with 10 µg/ml of each of the ADCs from Examples 179a, 226a, 85b, and 208b. After 72 hours, the cells were washed with PBS, lysed, and deep-frozen (-80°C). Using the methods described above, cell lysates and cell culture supernatants were post-processed and the following metabolites were identified and quantified after extraction:

.

C5b: Identification of ADC metabolites in vivo

After intravenous administration of 3–30 mg/kg of different ADCs, plasma and tumor concentrations of the ADC and possible metabolites can be measured and pharmacokinetic parameters such as clearance (CL), area under the curve (AUC), and half-life (t _1/2 ) can be calculated.

Assays for quantification of possible metabolites

Compounds were measured in plasma and tumors by high-pressure liquid chromatography (HPLC) coupled to triple quadrupole mass spectrometry (MS) after protein precipitation with methanol or acetonitrile.

For post-processing of 50 μL of plasma, add 250 μL of precipitation reagent (usually acetonitrile) and shake for 10 seconds. The precipitation reagent contains an internal standard (ISTD) at an appropriate concentration (usually 20-100 ng/mL). After centrifugation at 16,000 g for 3 minutes, transfer the supernatant to an autosampler vial, add 500 μL of the appropriate buffer for the eluent, and shake again.

During post-processing of the tumor, it was treated with 3 times the amount of extraction buffer. The extraction buffer contained 50 ml of tissue protein extractant (Pierce, Rockford, IL), two Complete-Protease-Inhibitor-Cocktail pellets (Roche Diagnostics GmbH, Mannheim, Germany) and phenylmethylsulfonyl fluoride (Sigma, St. Louis, MO) at a final concentration of 1 mM. The sample was homogenized twice for 20 minutes in a tissue grinder II (Qiagen) at maximum reciprocating speed. 50 microliters of the homogenate were transferred to an automatic sampling bottle and supplemented with 150 microliters of methanol containing ISTD. After centrifugation at 16,000 g for 3 minutes, 10 microliters of the supernatant were supplemented with 180 microliters of a buffer suitable for the eluent and re-vortexed. The tumor sample was then ready for measurement.

Finally both matrix samples were measured using HPLC coupled to a triple quadrupole mass spectrometer API 6500 from AB SCIEX Deutschland GmbH.

For calibration, plasma samples were spiked with concentrations ranging from 0.5 to 2000 µg/l. The limit of detection (LOQ) was approximately 2 µg/l. The linear range extended from 2 to 1000 µg/l.

To calibrate the tumor samples, concentrations of 0.5–200 µg/l were spiked into untreated tumor supernatant. The limit of detection was 5 µg/l. The linear range extended from 5 to 200 µg/l.

The quality controls used to test efficacy contained 5 and 50 µg/l and additionally 500 µg/l in plasma.

After administration of 10 mg/kg of ADCs from Examples 119b and 104b to a control group in a xenograft model with NCI-H292, mice were sacrificed 24 hours later, blood was drawn, and tumors isolated. Using the above-described method, plasma and tumor samples were post-processed and the following metabolites were identified and quantified after extraction:

.

Assays for quantification of antibodies used

The antibody content of the ADC was determined using a ligand binding assay (ELISA) based on the total IgG concentration in plasma samples and tumor lysates. A sandwich ELISA format was used. This ELISA has been validated and verified for use in both plasma and tumor samples. An ELISA plate was coated with a goat anti-human IgG Fc antibody. After incubation with the sample, the plate was washed and incubated with a simian anti-human IgG (H+L) antibody and a horseradish peroxidase (HRP) detection conjugate. After a further wash step, HRP substrate was added to the OPD, and color development was monitored by absorbance at 490 nm. A four-parameter equation was used to fit standard samples with known IgG concentrations. Unknown concentrations were determined by interpolation within the lower and upper limits of quantification (LLOQ).

C-6 in vivo efficacy test

For example, the efficacy of the conjugates of the present invention can be tested using a xenograft model. Those skilled in the art are familiar with prior art methods for testing the efficacy of the compounds of the present invention (see, for example, WO 2005/081711; Polson et al., Cancer Res. 2009 Mar 15;69(6):2358-64). To this end, a tumor cell line expressing the target molecule of the conjugate is transplanted into a rodent (e.g., a mouse). The conjugate of the present invention, an isotype antibody control conjugate, a control antibody, or isotonic saline is then administered to the transplanted animals. Administration is performed one or more times. After several days of incubation, tumor size is measured by comparing the animals treated with the conjugate to the control group. The animals treated with the conjugate exhibit smaller tumor size.

C-6a. Growth inhibition/regression of experimental tumors in mice

Human tumor cells expressing the antigen of the antibody-active substance conjugate are subcutaneously inoculated into the flank of immunosuppressed mice, such as NMR-inhibited nude mice or SCID mice. One to ten million cells are isolated from cell culture, centrifuged, and resuspended in culture medium or culture medium/Matrigel. This cell suspension is then injected subcutaneously into the mouse.

Over several days, the tumor grows. Treatment is initiated after the tumor is established (approximately 40 mm² in size). To test the effect on larger tumors, treatment may be initiated after the tumor has reached a size of 50-100 mm².

ADC treatment was administered via intravenous injection into the tail vein of mice. ADC was administered at a volume of 5 ml/kg.

The treatment schedule depends on the pharmacokinetics of the antibody. As a standard, treatment is given three times in a row every four days. For rapid assessment, a single treatment schedule can be used. However, this treatment can also be continued or can be followed later by a second three-treatment day cycle.

As a standard, 8 animals were used per treatment group. In addition to the group to which the active substance was administered, one group was treated as a control group using only buffer according to the same procedure.

During the experiment, tumor area was measured regularly in two dimensions (length/width) using calipers. Tumor area was determined as length x width. The mean tumor area ratio of the treated group to the control group was designated as T/C area.

When all experimental groups are terminated at the same time after the end of treatment, the tumors can be removed and weighed. The average tumor weight ratio of the treatment group to the control group is designated as T/C weight.

C-6b. Efficacy of Anti-EGFR Antibody-Active Agent Conjugates in the NCI-H292 Tumor Model Upon Repeat Treatment

One million NCI-H292 cells were inoculated subcutaneously into the flanks of female NMRI nude mice (Janvier). Treatment was initiated intravenously (Days 7, 11, and 15) at doses of 3 or 10 mg/kg when tumors reached 35 mm² on Day 7. Following treatment, tumor growth was monitored until Day 105.

Treatment with the antibody-active substance conjugates according to Examples 01a and 02a resulted in significant inhibition of tumor growth compared to vehicle and isotype controls. Initially, regardless of dose, most tumors regressed. Table 6 shows the T/C area values, measured by tumor area, at day 35. Treatment with the control conjugate (an isotype antibody directed against an irrelevant antigen) resulted in significantly weaker tumor growth inhibition. Treatment with the unconjugated antibody also resulted in growth inhibition; however, this was less severe than with the antibody-active substance conjugate .

C-6c. Efficacy of Anti-EGFR Antibody-Active Agent Conjugates in the NCI-H292 Tumor Model as a Single Treatment

One million NCI-H292 cells were inoculated subcutaneously into the flanks of female NMRI nude mice (Janvier). On day 9, when tumors reached a size of ~37 mm², mice were treated intravenously once with either 3 mg/kg of ADC or 2.5 mg/kg of cluster A (N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide) or cluster B (N-[(3R)-3-amino-4-fluorobutyl]-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide). Following this treatment, tumor growth was monitored until day 25.

Treatment with the antibody-active substance conjugates according to Examples 35A and 02A resulted in significant inhibition of tumor growth compared to the control group. During treatment with 3 mg/kg, most tumors regressed. Table 7 shows the T/C values for tumor weight and tumor area measured on day 25. Antibody-active substance conjugate 35A demonstrated superior efficacy compared to cetuximab and toxin clusters A or B.

C-6d. Efficacy of anti-EGFR and anti-TWEAKR antibody-active substance conjugates in the NCI-H292 tumor model as a single treatment

One million NCI-H292 cells were inoculated subcutaneously into the flanks of female NMRI nude mice (Janvier). On day 7, when tumors reached a size of 37 mm², mice were treated intravenously at a dose of 3 or 10 mg/kg of the antibody-active substance conjugate . Following this treatment, tumor growth was monitored until day 24.

A single treatment with anti-TWEAKR antibody active substance conjugate 02B resulted in significant and durable inhibition of tumor growth compared to the control group and unconjugated anti-TWEAKR antibody. Table 8 shows the T/C values measured on day 24 for tumor weight and tumor area.

C-6e. Efficacy of Anti-TWEAKR Antibody-Active Agent Conjugates in the BxPC3 Tumor Model Upon Multiple Treatments

Two million BxPC3 cells were inoculated subcutaneously into the flanks of female NMRI nude mice (Janvier). When tumors reached 45 mm² on day 10, treatment was initiated intravenously at a dose of 10 mg/kg (Days 10, 14, and 18). Following this treatment, tumor growth was monitored until Day 42.

Treatment with the antibody-active substance conjugates according to Examples 07B, 08B, 12B, 15B, and 46B resulted in significant inhibition of tumor growth compared to the control group. Table 9 shows the T/C values for tumor weight and tumor area measured on day 42. Treatment with the respective control conjugates (isotype antibodies against irrelevant antigens) resulted in significantly weaker tumor growth inhibition. Treatment with unconjugated antibodies also resulted in weaker tumor growth inhibition in some cases.

C-6f. Efficacy of anti-TWEAKR antibody-active substance conjugates in the NCI-H292 tumor model as a single treatment (2 independent experiments)

In both studies, 1 million NCI-H292 cells were inoculated subcutaneously into the flanks of female NMRI nude mice (Janvier). On day 10 (experiment 1) or day 8 (experiment 2), when tumors reached a size of ~45 mm², treatment was administered intravenously at a dose of 3 mg/kg of the antibody-active substance conjugate . Following treatment, tumor growth was monitored until day 18 (experiment 1) or day 24 (experiment 2).

In Experiment 1, single treatment with anti-TWEAKR antibody active substance conjugates 02B, 07B, and 08B resulted in significant inhibition of tumor growth compared to the control group and unconjugated anti-TWEAKR antibody. Table 10 (Experiment 1) shows the T/C values for tumor weight and tumor area measured on day 18. In Experiment 2, single treatment with anti-TWEAKR antibody active substance conjugates 155B, 173B, 165B, and 085B also resulted in significant inhibition of tumor growth compared to the control group and respective isotype controls (not shown). Table 11 (Experiment 2) shows the T/C values for tumor area measured on day 24.

C-6g. Efficacy of anti-TWEAKR antibody-active substance conjugates in the A375 tumor model upon multiple treatments

Five million A375 (human melanoma) cells were inoculated subcutaneously into the flanks of female NMRI nude mice (Janvier). Treatment was initiated intravenously (Days 10, 14, and 18) at a dose of 10 mg/kg when tumors reached 41 mm² on Day 10. Following this treatment, tumor growth was monitored until Day 22.

Treatment with the antibody-active substance conjugates according to Examples 38B and 104B resulted in significant inhibition of tumor growth compared to the control group. Table 10 shows the T/C values for tumor weight and tumor area measured on day 22. Treatment with the respective control conjugates (isotype antibodies against irrelevant antigens) resulted in significantly weaker tumor growth inhibition. Treatment with unconjugated antibodies also resulted in weaker tumor growth inhibition in some cases.

C-6h. Efficacy of anti-TWEAKR antibody-active substance conjugates in the LoVo tumor model upon multiple treatments

Five million LoVo (human colon cancer) cells were inoculated subcutaneously into the flanks of female NMRI nude mice (Janvier). Treatment was initiated intravenously (Days 7, 11, and 15) at a dose of 10 mg/kg when tumors reached 43 mm² on Day 7. Following this treatment, tumor growth was monitored until Day 45.

Treatment with the antibody-active substance conjugates according to Examples 07B, 87B, and 104B resulted in significant inhibition of tumor growth compared to the control group. Table 11 shows the T/C values for tumor weight and tumor area measured on day 45. Treatment with the control conjugate of 07B (an antibody of the same type directed against an irrelevant antigen) resulted in significantly weaker inhibition of tumor growth.

D. Examples of Pharmaceutical Compositions

The compounds of the present invention can be converted into pharmaceutical preparations as follows:

Intravenous solution:

The compound of the invention is dissolved at a concentration below saturation solubility in a physiologically acceptable solvent (e.g., isotonic saline solution, D-PBS, or a formulation containing glycine and sodium chloride in citrate buffer supplemented with polysorbate 80). The solution is sterile filtered and dispensed into sterile and pyrogen-free injection containers.

Intravenous solution:

The compounds according to the invention can be converted into the administration forms mentioned. This can be achieved in a manner known per se by "mixing" with or "dissolving" in inert, non-toxic, pharmaceutically acceptable adjuvants (e.g. buffer substances, stabilizers, solubilizers, preservatives). It may contain, for example, the following: amino acids (glycine, histidine, methionine, arginine, lysine, leucine, isoleucine, threonine, glutamic acid, phenylalanine, etc.), sugars and related compounds (glucose, sucrose, mannitol, trehalose, sucrose, mannose, lactose, sorbitol), glycerol, sodium, potassium, ammonium and calcium salts (e.g., sodium chloride, potassium chloride or disodium hydrogen phosphate and many others), acetate/acetic acid buffer systems, phosphate buffer systems, citric acid and citrate buffer systems, tromethamine (TRIS and TRIS salts), polysorbates (e.g., polysorbate 80 and polysorbate 20), poloxamers (e.g., poloxamer 188 and poloxamer 171), polyethylene glycol (PEG derivatives, e.g., 3350), Triton X-100, EDTA salts, glutathione, albumin (e.g., human), urea, benzyl alcohol, phenol, chlorocresol, m-cresol, benzalkonium chloride and many others.

Lyophilized product for subsequent conversion into intravenous, subcutaneous or intramuscular solutions:

Alternatively, the compound of the present invention can be converted into a stable lyophilized product (possibly with the aid of the above-mentioned adjuvants) and reconstituted with a suitable solvent (eg, injection-grade water, isotonic saline solution) before administration and administered.

Examples of anti-TWEAKR antibodies

Unless otherwise described in detail herein, all examples were performed using standard methods known to those skilled in the art. Conventional methods of molecular biology for the following examples can be performed as described in standard laboratory textbooks, such as Sambrook et al., Molecular Cloning: a Laboratory Manual, 2nd ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

AK Example 1: Antibody Preparation Using Antibody Library

The TWEAKR-specific human monoclonal antibodies of the present invention (Hoogenboom H.R., Nat Biotechnol 2005; 23(3): 1105-16) were isolated by protein screening using a whole human phage display library (Hoet RM et al., Nat Biotechnol 2005; 23(3): 344-8) in which the dimeric Fc-fused extracellular domains of human and murine TWEAKR were immobilized as targets.

The antigen was biotinylated using approximately a 2-fold molar excess of biotin-LC-NHS (Pierce; catalog no. 21347) according to the manufacturer's instructions and desalted using Zeba Desalting Columns (Pierce; catalog no. 89889). Washed magnetic beads (DynaBeads) were incubated with 200 nM biotinylated human antigen overnight at 4°C and blocked with blocking buffer (3% BSA, 0.05% Tween-20 in PBS) for 1 hour at 4°C. The blocked Fab phage library was added to the blocked TWEAKR beads (DynaBeads Streptavidin-M280 - Invitrogen 112-06D) and incubated at room temperature for 30 minutes. After stringent washing (3 x with blocking buffer and 9 x with PBS (150 mM NaCl; 8 mM Na2HPO4; 1.5 mM KH2PO4; adjusted to pH = 7.4-7.6), containing 0.05% Tween-20), Fab phages specifically bound to biotinylated TWEAKR beads (DynaBeads Streptavidin-M280 - Invitrogen 112-06D) were resuspended in PBS and used directly to infect E. coli strain TG1 for amplification. In the second selection round, both mouse TWEAKRs were used (200 nM) to select for cross-reactive binders, and in the third selection round, the concentration of human TWEAKR was reduced (100 nM) to increase the selection pressure for high-affinity binders.

11 different Fab phages were identified and the corresponding antibodies were cloned into mammalian IgG expression vectors that provide a missing CH2-CH3 domain that is not present in soluble FAbs. The resulting IgG was transiently expressed in mammalian cells as described in Tom et al., Chapter 12: Expression Systems in Methods Express, edited by Michael R. Dyson and Yves Durocher, Scion Publishing Ltd, 2007. Briefly, expression plasmids based on the CMV promoter were transfected into HEK293-6E cells and cultured in Fernbach bottles or Wave bags. Expression was performed at 37°C for 5 to 6 days in F17 medium (Invitrogen). 1% Ultra-Low IgG FCS (Invitrogen) and 0.5 mM valproic acid (Sigma) were added as supplements 24 hours after transfection. The antibodies were purified by protein-A chromatography and further characterized by their binding affinity to soluble monomeric TWEAKR using ELISA and BIAcore analysis as described in AK-Example 2.

To determine the cellular binding characteristics of anti-TWEAKR antibodies, binding to various cell lines (HT29, HS68, and HS578) was examined by flow cytometry. Cells were suspended in a dilution of the antibody (5 µg/ml) in FACS buffer and incubated on ice for 1 hour. Secondary antibody (PE goat anti-human IgG, Dianova #109-115-098) was then added. After incubation on ice for 1 hour, cells were analyzed by flow cytometry using a FACS array (BD Biosciences).

An NF-κB reporter gene assay was performed to assess the agonistic activity of all 11 identified antibodies (human IgG1). HEK293 cells were transiently transfected with an NF-κB reporter gene construct (BioCat, catalog number LR-0051-PA) using 293fectin according to the manufacturer's instructions. Transfected cells were seeded in white poly-lysine-coated 384-well plates (BD) in F17 medium (serum-free; Invitrogen) at 37°C and 5% CO2. The next day, the cells were stimulated with various concentrations of purified antibodies for 6 hours, followed by a luciferase assay using standard methods.

Internalization was monitored via fluorescent labeling with an anti-TWEAKR antibody (CypHer 5E monoNHS ester; GE Healthcare). Before treatment, HT29 cells (2 x 10 ^⁴ /well) were seeded in 100 μl of culture medium in 96-well MTP plates (thick, black, clear bottom, No. 4308776, Applied Biosystems). After 18 hours of incubation at 37°C/5% CO _₂ , the medium was replaced and labeled anti-TWEAKR antibody was added at various concentrations (10, 5, 2.5, 1, and 0.1 μg/ml). Incubation times were 0, 0.25, 0.5, 1, 1.5, 2, 3, 6, and 24 hours. Fluorescence measurements were performed on an InCell Analyser 1000 (GE Healthcare).

The antibody with the highest in vitro potency (TPP-883) was selected for further potency and affinity maturation.

Amino acid sequences of the light chain (SEQ ID NO. 71) and heavy chain (SEQ ID NO. 72) of TPP-883; the CDRs of the heavy and light chains are underlined.

Maturation was performed in a first mutation collection round, followed by recombination of those amino acid modifications that most improved affinity and potency. To collect mutant NNK (N = AGCT, K = G or T), randomization was performed at the following independent amino acid positions by site-directed mutagenesis using synthetic oligonucleotides encompassing NNK codon diversification (consecutive amino acid designations, see Figure 25): S35, S36, Y37, and N39 in CDR-L1; A51, S53, S54, Q56, and S57 in CDR-L2; S92, Y93, S94, S95, G97, and I98 in CDR-L3; P31, Y32, P33, M34, and M35 in CDR-H1; Y50, S52, P53, S54, G56, K57, and H59 in CDR-H2; and G99, G100, D101, G102, Y103, F104, D105, and Y106 in CDR-H3. DNA from all independent NNK saturation mutagenesis libraries was cloned into a mammalian IgG expression vector for potency maturation or into a phagemid vector for affinity maturation. Affinity maturation was performed by phage selection. Washed magnetic beads (DynaBeads) were incubated with 10 nM, 1 nM, 100 pM, or 10 pM biotinylated human antigen overnight at 4°C and blocked with blocking buffer (3% BSA, 0.05% Tween-20 in PBS) for 1 hour at 4°C. The blocked Fab phage pool was added to the blocked TWEAKR-DynaBeads at 10,000-fold, 1,000-fold, and 100-fold excesses compared to the theoretical pool complexity (Bibliothekkomplexität) and incubated at room temperature for 30 minutes. This means that a total of 12 strategies (4 antigen concentrations x 3 Fab phage titers) were followed. After stringent washing (3 times with blocking buffer and 9 times with PBS containing 0.05% Tween-20), Fab phage specifically bound to biotinylated TWEAKR DynaBeads (DynaBeads Streptavidin-M280 - Invitrogen 112-06D) were resuspended in PBS and used directly to infect E. coli strain TG1 for amplification. In selection round 2, the concentration of human TWEAKR-Fc was reduced (1 nM, 100 pM, 10 pM, and 1 pM), and the same Fab phage titer (4.4 x 10 ¹¹ ) was used for all 12 strategies. To express soluble Fab, the phagemid vector was digested with MluI to remove the gene-III membrane anchor sequence required for Fab display on phage and religated. 96 variants from each of the 12 selection pools were expressed as soluble Fab and tested in an ELISA format. To this end, 2.5 nM biotinylated TWEAKR-Fc was antigen-coated, and binding of the soluble Fab was verified using an anti-c-Myc antibody (Abcam ab62928). Seven single-substitution variants (consecutive amino acid designations, see Figure 25 ) were validated for improved binding to TWEAKR-Fc (Seq ID No. 138): S36G in CDR-L1, A51Q and S57K in CDR-L2, S94T and G97F in CDR-L3, M35I in CDR-H1, and G102T in CDR-H3. For potency maturation, HEK293 cells were transfected with an NF-κB reporter gene (BioCat, catalog number LR-0051-PA). Transfected cells were seeded in F17 medium (serum-free; Invitrogen) onto white polylysine-coated 384-well plates (BD) and transiently expressed in mammalian cells from the NNK diverse positional antibody (human IgG1) library of independent variants. The following day, NF-κB reporter cells were stimulated with the expressed independent NNK antibody variants for 6 hours, followed by luciferase assays using standard methods. One single-substitution variant with improved agonistic activity was detected: G102T in CDR-H3. This variant was also obtained through affinity maturation and also exhibited the highest affinity enhancement. After collecting mutations through affinity and potency screening, all seven favorable single substitutions (library complexity: 128 variants) were recombined into a recombinant library. To this end, oligonucleotides were synthesized to introduce the selected mutation or the corresponding wild-type amino acid at each selected position. The library was constructed using sequential rounds of overlap extension PCR. The final PCR product was ligated into a bacterial soluble Fab expression vector, and 528 variants were randomly selected (~4-fold excess of the sample taken) for equilibrium ELISA screening using soluble Fab as described above. Finally, 7 variants were selected based on their improved affinity compared to the best single-substitution variant G102T. These corresponding DNAs were themselves cloned into a mammalian IgG expression vector and tested for functional activity in the NF-κB reporter gene cell assay mentioned above. Finally, the resulting sequences were compared with human germline sequences and any deviations that did not significantly affect affinity and potency were adjusted. Antibodies with the following sequences were obtained through antibody library screening and affinity and/or potency maturation:

Amino acid sequences of the light chain (SEQ ID NO. 1) and heavy chain (SEQ ID NO. 2) of TPP-2090; the CDRs of the heavy and light chains are underlined.

Amino acid sequences of the light chain (SEQ ID NO. 11) and heavy chain (SEQ ID NO. 12) of TPP-2149; the CDRs of the heavy and light chains are underlined.

Amino acid sequences of the light chain (SEQ ID NO. 21) and heavy chain (SEQ ID NO. 22) of TPP-2093; the CDRs of the heavy and light chains are underlined.

Amino acid sequences of the light chain (SEQ ID NO. 31) and heavy chain (SEQ ID NO. 32) of TPP-2148; the CDRs of the heavy and light chains are underlined.

Amino acid sequences of the light chain (SEQ ID NO. 41) and heavy chain (SEQ ID NO. 42) of TPP-2084; the CDRs of the heavy and light chains are underlined.

Amino acid sequences of the light chain (SEQ ID NO. 51) and heavy chain (SEQ ID NO. 52) of TPP-2077; the CDRs of the heavy and light chains are underlined.

Amino acid sequences of the light chain (SEQ ID NO. 61) and heavy chain (SEQ ID NO. 62) of TPP-1538; the CDRs of the heavy and light chains are underlined.

Amino acid sequences of the light chain (SEQ ID NO. 81) and heavy chain (SEQ ID NO. 82) of TPP-1854; the CDRs of the heavy and light chains are underlined.

Amino acid sequences of the light chain (SEQ ID NO. 91) and heavy chain (SEQ ID NO. 92) of TPP-1853; the CDRs of the heavy and light chains are underlined.

Amino acid sequences of the light chain (SEQ ID NO. 101) and heavy chain (SEQ ID NO. 102) of TPP-1857; the CDRs of the heavy and light chains are underlined.

Amino acid sequences of the light chain (SEQ ID NO. 111) and heavy chain (SEQ ID NO. 112) of TPP-1858; the CDRs of the heavy and light chains are underlined.

Example 2: Biochemical characterization of antibodies

Binding affinity was determined by Biacore analysis:

The binding affinity of the anti-TWEAKR antibody was examined using surface plasmon resonance analysis on a Biacore T100 instrument (GE Healthcare Biacore, Inc.). The antibody was immobilized on a CM5 sensor chip using an indirect capture reagent, anti-human IgG (Fc). Reagents from the "Human Antibody Capture Kit" (BR-1008-39, GE Healthcare Biacore, Inc.) were used as described by the manufacturer. A 10 µg/ml concentration of anti-TWEAKR antibody was injected at 10 µl/min for 10 seconds.

Purified recombinant human TWEAKR protein (TPP-2305, SEQ ID NO:168) at various concentrations (200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.12 nM, and 1.56 nM) in HEPES-EP buffer (GE Healthcare Biacore, Inc.) was injected over the immobilized anti-TWEAKR antibody at a flow rate of 60 µl/min for 3 minutes, with a dissociation time of 5 minutes. Sensorgrams were generated after online reference cell correction, followed by subtraction of the buffer sample. The dissociation constant ( _KD ) was calculated based on the ratio of the association constant ( _kon ) and dissociation constant ( _koff ) obtained by fitting the sensorgrams using a 1:1 first-order binding model.

The antibodies of the present invention were determined to bind to TWEAKR with moderate affinity (K _D 10-200 nM), while some comparative antibodies (e.g., PDL-192(TPP-1104), 136.1(TPP-2194), 18.3.3(TPP-2193), P4A8(TPP-1324), P3G5(TPP-2195), P2D3(TPP-2196), ITEM-1, ITEM-4) exhibited high affinity binding (0.7-3.7 nM). The sequences of the variable domains of antibodies PDL-192, 136.1, 18.3.3, P4A8, P3G5 and P2D3 were obtained from patent documents WO2009/020933 and WO2009/140177, and the coding sequences of the constant regions of human IgG1 and mouse IgG2 were added to produce full-length IgG PDL-192 (TPP-1104), 136.1 (TPP-2194), 18.3.3 (TPP-2193), P4A8 (TPP-1324), P3G5 (TPP-2195), P2D3 (TPP-2196). The affinity ranges measured in this study agree well with published data: KD values of 5.5, 0.2, and 0.7 nM have been published for PDL-192, 18.3.3, and 136.1 (WO2009/020933); and 2.6 nM for P4A8 (WO2009/140177). For comparison, the natural ligand TWEAK binds to TWEAKR with a _KD value of 0.8–2.4 nM (Immunity. 2001 Nov;15(5):837-46; Biochem J. 2006 Jul 15;397(2):297-304; Arterioscler Thromb Vasc Biol. 2003 Apr 1;23(4):594-600).

Thus, it can be noted that as a result the antibodies of the invention (TPP-883, TPP-1538, TPP-2077, TPP-2084, TPP-2148, TPP-2093, TPP-2149 and TPP-2090) bind to TWEAKR with moderate affinity (K _D 10 - 200 nM).

Characterization of the binding epitope of TPP-2090 using N- and C-terminal truncation variants of the TWEAKR ectodomain:

An alignment of the cysteine-rich domain (amino acids 34-68) of TWEAKR from different species (Figure 1 - Alignment) shows that it is highly conserved across all six species analyzed. PDL-192 relies on R56 for binding (WO2009/020933: Figure 2B) and therefore does not bind to rat, porcine, or mouse TWEAKR. TPP-2090 relies on the conserved amino acid D47 for binding and therefore binds to all species shown.

In the first approach to characterize the binding epitopes of the antibodies mentioned above, N- and C-terminal truncation variants of the TWEAKR extracellular domain were generated and examined for their ability to bind to various anti-TWEAKR antibodies. Amino acids 28 to 33 were deleted at the N-terminus, and amino acids 69 to 80 were deleted at the C-terminus, leaving the cysteine-rich domain with disulfide bridges between Cys36-Cys49, Cys52-Cys67, and Cys55-Cys64 intact (see Figure 2). Both constructs—the complete extracellular domain 28-80 and the truncated extracellular domain 34-68, including both the N- and C-termini—were expressed and purified as Fc fusion proteins, TPP-2202 and TPP-2203.

To analyze binding, 1 µg/ml of the corresponding dimeric TWEAKR Fc construct was applied, and 0.3 µg/ml and 0.08 µg/ml of biotinylated IgG were used as soluble binding partners. Detection was performed using streptavidin-HRP and Amplex Red substrate. IgG was biotinylated with an approximately 2-fold molar excess of biotin-LC-NHS (Pierce; Catalog No. 21347) according to the manufacturer's instructions and desalted using Zeba Desalting Columns (Pierce; Catalog No. 89889). The antibodies of the present invention exhibited saturated binding to both constructs at all concentrations of soluble ligand used, while antibodies P4A8 (TPP-1324), P3G5 (TPP-2195), and Item-4 exhibited saturated binding only to the full-length extracellular domain, with reduced binding to the N- and C-terminally truncated constructs (Figures 3 & 4). This suggests that the binding epitope of the antibodies of the present invention is located within the cysteine-rich domain between amino acids 34-68. To analyze whether P4A8 (TPP-1324) and P3G5 (TPP-2195) binding requires the N-terminus or C-terminus of the TWEAKR ectodomain, monomeric ectodomains were generated with a C-terminal deletion of amino acids 69 to 80. Binding of P4A8 (TPP-1324) and P3G5 (TPP-2195) to the C-terminally truncated TWEAKR ectodomain was similarly impaired, whereas the antibodies of the present invention exhibited saturated binding ( Figure 5 ).

Thus, the binding epitopes of TPP-2090, TPP-2084, PDL-192 (TPP-1104) and 136.1 (TPP-2194) in the cysteine-rich domain and the binding epitopes of P4A8 (TPP-1324) and P3G5 (TPP-2195) are at least partially located outside the cysteine-rich domain.

Effect of TWEAKR-Fc mutant protein on antibody affinity

To examine the binding characteristics of the antibodies of the present invention in more detail, certain mutant proteins of TWEAKR that have been proposed to be associated with the effects of known agonistic antibodies (WO2009/140177) were investigated. To this end, the full-length extracellular domain (amino acids 28-80) with the following individual amino acid substitutions was expressed and purified as an Fc fusion protein: T33Q; S40R; W42A; M50A; R56P; H60K; L65Q.

To obtain dose-response data, different TWEAKR-Fc mutant proteins were coated onto 384-well Maxisorb ELISA plates at low concentrations (62 ng/ml) and serial two-fold dilutions of biotinylated IgG starting at a concentration of 100 nM were used as soluble binding partners. Detection was performed using streptavidin-HRP and Amplex Red. The IgGs examined were TPP-2090 and TPP-2084 of the present invention, PDL-192, 136.1, and 18.3.3 of WO2009/020933, P4A8 and P3G5 of WO2009/140177, and ITEM-1 and ITEM-4 of Nakayama et al. [Biochem Biophys Res Com 306: 819-825].

IgG was biotinylated using approximately a 2-fold molar excess of biotin-LC-NHS (Pierce; Catalog No. 21347) according to the manufacturer's instructions and desalted using Zeba Desalting Columns (Pierce; Catalog No. 89889). The dose-response data were fitted and the IC50 values were determined. To illustrate the results, a table was generated; "-" marks IC50 values above 50 nM and "+" marks IC50 values between 1 and 150 pM.

As previously reported, ITEM-4 exhibited poor binding to the H60K mutant protein [WO2009/140177: FIG23F], and PDL-192 exhibited poor binding to the R56P mutant protein [WO2009/020933: FIG22B]. Unlike the published data, ITEM-1 exhibited poor binding to R56P, and all antibodies exhibited poor binding to W42A [WO2009/140177: FIG23E, FIG23F]. These differences may be explained by the chosen method.

Unlike ITEM-1, ITEM-4, PDL-192, 136.1, and 18.3.3, the antibodies of the present invention bind regardless of all substitutions except W42A.

Alanine scanning of cysteine-rich domains:

Alanine scanning of the cysteine-rich domain (amino acids 34-68) was performed to determine the binding site of the antibodies of the invention. Figure 6 demonstrates that N- and C-terminal truncation variants of the full-length extracellular domain of TWEAKR do not impair binding of the antibodies of the invention. Thus, the binding epitope is located in the cysteine-rich domain. The following substitutions were introduced into the TWEAKR (34-68) Fc construct: S37A, R38A, S40A, S41A, W42A, S43A, D45A, D47A, K48A, D51A, S54A, R56A, R58A, P59A, H60A, S61A, D62A, F63A, and L65A.

These TWEAKR (34-68) Fc mutants were expressed in HEK293 cells. To obtain dose-response data, IgG was coated onto 384-well Maxisorp ELISA plates at a concentration of 1 μg/ml and serial two-fold dilutions of supernatant containing TWEAKR mutants were used as soluble binding partners. Detection was performed using anti-HIS-HRP and Amplex Red. The IgGs tested were TPP-2090 of the present invention, PDL-192 of WO2009/020933, and P4A8 of WO2009/140177.

To assess the relevance of TWEAKR muteins for binding to various IgGs, blots were prepared at specific mutein concentrations. For example, Figure 6 shows a blot of 8-fold diluted supernatants from TWEAKR expression broth, with PDL-192 (TPP-1104) on the X-axis and TPP-2090 on the Y-axis. This blot demonstrates that the substitution D47A impairs binding to TPP-2090, while the substitution R56A impairs binding to PDL-192 (TPP-1104). None of these constructs demonstrated binding to P4A8 (TPP-1324), consistent with the results obtained above (Figure 6). Therefore, the P4A8 epitope is at least partially located outside the cysteine-rich domain.

The dependence of antibody interactions on the recognition of certain TWEAKR amino acids correlated with the agonist activity measured for these antibodies. The natural ligand, TWEAK, exhibits potent activation of TWEAKR and is dependent on leucine 46 for binding to the cysteine-rich domain of TWEAKR (Pellegrini et al., FEBS 280:1818-1829). P4A8 exhibits very low agonist activity and interacts, at least in part, with domains outside the cysteine-rich domain of TWEAKR. PDL-192 exhibits moderate agonist activity and is dependent on R56 for binding to the cysteine-rich domain, but opposite the TWEAK ligand site. TPP-2090 and TWEAK are dependent on D47 and L46 for binding, and therefore bind to similar binding sites (Figure 7).

It can be concluded that the antibodies of the present invention (eg, TPP-2090) bind to TWEAKR in a D47-dependent manner.

The dependence of antibody interactions on the recognition of certain TWEAKR amino acids correlates with the agonist activity measured for these antibodies. The natural ligand, TWEAK, exhibits potent activation of TWEAKR and is dependent on leucine 46 for binding to the cysteine-rich domain of TWEAKR (Pellegrini et al., FEBS 280:1818-1829). P4A8 exhibits very low agonist activity and interacts, at least in part, with domains outside the cysteine-rich domain of TWEAKR. PDL-192 exhibits moderate agonist activity and is dependent on R56 for binding to the cysteine-rich domain, but opposite the TWEAK ligand site. The antibodies of the present invention (e.g., TPP-2090) are dependent on D47 for binding, while TWEAK is dependent on L46 for binding, but binds to a similar but distinct binding site (Figure 7). Thus, the antibodies of the present invention that exhibit strong agonist activity bind to a novel epitope (D47 dependence) associated with very high agonist activity. Interestingly, Michaelson et al. (see page 369, left column, Michaelson JS et al., MAbs. 2011 Jul-Aug; 3(4): 362-75) provide an explanation for why all agonistic antibodies they examined had weaker agonistic activity than the natural ligand, TWEAK. They conclude that the reduced potency may be due to the antibody's dimeric binding interaction with TWEAKR, which may undergo a trimerization interaction. Thus, the surprising result is that the antibodies of the present invention have even higher agonistic activity despite their dimeric interaction with TWEAKR. This surprising effect is related to the specific binding properties of the antibodies of the present invention, namely, specific binding to D47 of TWEAKR.

Further implementation plan

1. A conjugate of a binding body or a derivative thereof and one or more active substance molecules, wherein the active substance molecule is a spindle kinesin inhibitor connected to the binding body via a linker L.

2. The conjugate according to item 1, wherein the binder or its derivative is a binder peptide or protein or a derivative of a binder peptide or protein.

3. The conjugate according to item 2, wherein each active substance molecule is linked to different amino acids of the conjugate peptide or protein or its derivative via a linker.

4. The conjugate according to one or more of the preceding items, wherein the conjugate has an average of 1.2 to 20 molecules of active substance per binding body.

5. Conjugate according to one or more of items 2 to 4, wherein the binding peptide or protein represents an antibody or a derivative of the binding peptide or protein.

6. The conjugate according to one or more of the preceding items, wherein the binding entity binds to a cancer target molecule.

7. The conjugate according to item 6, wherein the binding entity binds to an extracellular target molecule.

8. The conjugate according to item 7, wherein the binding body is internalized by cells expressing the target molecule after binding to the extracellular target molecule and processed within the cell (preferably via lysosomes).

9. The conjugate according to one or more of items 2 to 8, wherein the binding peptide or protein is a human, humanized or chimeric monoclonal antibody or an antigen-binding fragment thereof.

10. The conjugate according to item 9, wherein the binding peptide or protein is an anti-HER2 antibody, an anti-EGFR antibody, an anti-TWEAKR antibody or an antigen-binding fragment thereof.

11. The conjugate according to item 10, wherein the anti-TWEAKR antibody specifically binds to amino acid D (D47) in position 47 of TWEAKR (SEQ ID NO: 169), preferably the anti-TWEAKR antibody TPP-2090.

12. The conjugate according to one or more of the preceding items, wherein the spindle kinesin inhibitor has the following substructure and salts, solvates and salts of solvates thereof:

in

#a represents the bond to the rest of the molecule;

R ^1a represents H or -(CH ₂ ) _0-3 Z, wherein Z represents -H, -OY ³ , -SY ³ , -NHY ³ , halogen, -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently of one another represent H, NH ₂ , -(CH ₂ CH ₂ O) _0-3 -(CH ₂ ) _0-3 Z' or -CH(CH ₂ W)Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ , COOH, -NH-CO-CH ₂ -CH ₂ -CH(NH ₂ )COOH or -(CO-NH-CHY ⁴ ) _1-3 COOH, wherein W represents H or OH, wherein Y ⁴ independently of one another represent linear or branched C _1-6 -alkyl optionally substituted by -NHCONH ₂ , or represent aryl or benzyl optionally substituted by -NH ₂ ;

R ^2a and R ^4a independently represent H, -CO-CHY ⁴ -NHY ⁵ or -(CH ₂ ) _0-3 Z, or R ^2a and R ^4a together (forming a pyrrolidine ring) represent -CH ₂ -CHR ¹⁰ - or -CHR ¹⁰ -CH ₂ -, wherein R ¹⁰ represents H, NH ₂ , COOH, SO ₃ H, SH or OH,

wherein Z represents -H, -OY ³ , -SY ³ , -NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH;

wherein Y ⁴ independently of one another represents linear or branched C _1-6 alkyl optionally substituted by -NHCONH ₂ , or represents aryl or benzyl optionally substituted by -NH ₂ , and Y ⁵ represents H or -CO-CHY ⁶ -NH ₂ , wherein Y ⁶ represents linear or branched C _1-6 -alkyl;

The spindle kinesin inhibitor is connected to the linker by replacing a hydrogen atom at R ^1a , R ^2a , R ^4a or a hydrogen atom at the pyrrolidine ring formed by R ^2a and R ^4a .

13. The conjugate according to one or more of the preceding items, wherein the spindle kinesin inhibitor is represented by the general formula (I) and its salts, solvates and salts of solvates:

in

R ^1a represents H or -(CH ₂ ) _0-3 Z, wherein Z represents -H, -OY ³ , -SY ³ , -NHY ³ , halogen, -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently of one another represent H, NH ₂ , -(CH ₂ CH ₂ O) _0-3 -(CH ₂ ) _0-3 Z' or -CH(CH ₂ W)Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ , COOH, -NH-CO-CH ₂ -CH ₂ -CH(NH ₂ )COOH or -(CO-NH-CHY ⁴ ) _1-3 COOH, wherein W represents H or OH, wherein Y ⁴ independently of one another represent linear or branched C _1-6 -alkyl optionally substituted by -NHCONH ₂ , or represent aryl or benzyl optionally substituted by -NH ₂ ;

R ^2a and R ^4a independently represent H, -CO-CHY ⁴ -NHY ⁵ or -(CH ₂ ) _0-3 Z, or R ^2a and R ^4a together (forming a pyrrolidine ring) represent -CH ₂ -CHR ¹⁰ - or -CHR ¹⁰ -CH ₂ -, wherein R ¹⁰ represents H, SO ₃ H, NH ₂ , COOH, SH or OH,

wherein Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH;

wherein Y ⁴ independently of one another represents linear or branched C _1-6 alkyl optionally substituted by -NHCONH ₂ , or represents aryl or benzyl optionally substituted by -NH ₂ , and Y ⁵ represents H or -CO-CHY ⁶ -NH ₂ , wherein Y ⁶ represents linear or branched C _1-6 -alkyl;

R ^3a represents an optionally substituted alkyl, aryl, heteroaryl, heteroalkyl or heterocycloalkyl, preferably a C _1-10 -alkyl, C _6-10 -aryl or C _6-10 -aralkyl, C 5-10 -heteroalkyl, C _1-10 -alkyl-OC _6-10 -aryl- or C _5-10 -heterocycloalkyl, which may be substituted by 1-3 -OH groups, _1-3 halogen atoms, 1-3 haloalkyl groups (each of which may have 1-3 halogen atoms), 1-3 O-alkyl groups, 1-3 -SH groups, 1-3 -S-alkyl groups, 1-3 -O-CO-alkyl groups, 1-3 -O-CO-NH-alkyl groups, 1-3 -NH-CO-alkyl groups, 1-3 -NH-CO-NH-alkyl groups, 1-3 -S(O) _n -alkyl groups, 1-3 -SO ₂ -NH-alkyl, 1-3 -NH-alkyl, 1-3 -N(alkyl) ₂ groups, 1-3 -NH ₂ groups or 1-3 -(CH ₂ ) _0-3 Z groups, where Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ , where Y ¹ and Y ² independently of one another represent H, NH ₂ or -(CH ₂ ) _0-3 Z ', and Y ³ represents H, -(CH ₂ ) _0-3 -CH(NHCOCH ₃ )Z ', -(CH ₂ ) _0-3 -CH(NH ₂ )Z ' or -(CH ₂ ) _0-3 Z ', where Z ' represents H, SO ₃ H, NH ₂ or COOH (wherein "alkyl" preferably represents C _1-10 -alkyl);

R ^8a represents a C _1-10 -alkyl group;

HZ represents a monocyclic or bicyclic heterocycle which may be substituted by one or more substituents selected from halogen, C _1-10 -alkyl which may be optionally substituted by halogen, C _6-10 -aryl and C _6-10 -aralkyl;

The spindle kinesin inhibitor is connected to the linker by replacing a hydrogen atom at R ^1a , R ^2a , R ^3a , R ^4a , R ^8a or at the pyrrolidine ring formed by R ^2a and R ^4a .

14. The conjugate according to one or more of the preceding items, wherein the active substance molecule-linker is represented by the general formula (II) and its salts, solvates and salts of solvates:

in

_X1 represents N, _X2 represents N and _X3 represents C; or

_X1 represents N, _X2 represents C and _X3 represents N; or

_X1 represents CH, _X2 represents C and _X3 represents N; or

_X1 represents NH, _X2 represents C and _X3 represents C; or

_X1 represents CH, _X2 represents N and _X3 represents C;

R ¹ represents H, -L-#1 or -(CH ₂ ) _0-3 Z, wherein Z represents -H, -NHY ³ , -OY ³ , -SY ³ , halogen, -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently of one another represent H, NH ₂ , -(CH ₂ CH ₂ O) _0-3 -(CH ₂ ) _0-3 Z' or -CH(CH ₂ W)Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, NH ₂ , SO ₃ H, COOH, -NH-CO-CH ₂ -CH ₂ -CH(NH ₂ )COOH or -(CO-NH-CHY ⁴ ) _1-3 COOH, wherein W represents H or OH, wherein Y ⁴ independently of one another represent linear or branched C _1-6 -alkyl optionally substituted by -NHCONH ₂ , or represent aryl or benzyl optionally substituted by -NH ₂ ;

R ² and R ⁴ independently represent –L-#1, H, –CO-CHY ⁴ –NHY ⁵ or –(CH ₂ ) _0-3 Z, or R ² and R ⁴ together (forming a pyrrolidine ring) represent –CH ₂ -CHR ¹⁰ - or –CHR ¹⁰ -CH ₂ -, wherein R ¹⁰ represents L-#1, H, NH ₂ , SO ₃ H, COOH, SH or OH,

wherein Z represents -H, OY ³ , -SY ³ , halogen, NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH;

wherein Y ⁴ independently of one another represents linear or branched C _1-6 alkyl optionally substituted by -NHCONH ₂ , or represents aryl or benzyl optionally substituted by -NH ₂ , and Y ⁵ represents H or -CO-CHY ⁶ -NH ₂ , wherein Y ⁶ represents linear or branched C _1-6 -alkyl;

A represents CO, SO, SO ₂ , SO ₂ NH or CNNH;

R ³ represents –L-#1 or an optionally substituted alkyl, aryl, heteroaryl, heteroalkyl or heterocycloalkyl group, preferably –L-#1 or C _1-10 -alkyl, C _6-10 -aryl, C _6-10 -aralkyl, C _5-10 -heteroalkyl, C _1-10 -alkyl-OC _6-10 -aryl- or C _5-10 -heterocycloalkyl group, which may be substituted by 1-3 –OH groups, 1-3 halogen atoms, 1-3 haloalkyl groups (each of which may have 1-3 halogen atoms), 1-3 O-alkyl groups, 1-3 –SH groups, 1-3 -S-alkyl groups, 1-3 -O-CO-alkyl groups, 1-3 -O-CO-NH-alkyl groups, 1-3 -NH-CO-alkyl groups, 1-3 -NH-CO-NH-alkyl groups, 1-3 -S(O) _n -alkyl groups, 1-3 -SO ₂ -NH-alkyl, 1-3 -NH-alkyl, 1-3 -N(alkyl) ₂ groups, 1-3 -NH ₂ groups or 1-3 -(CH ₂ ) _0-3 Z groups, where Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ , where Y ¹ and Y ² independently of one another represent H, NH ₂ or -(CH ₂ ) _0-3 Z′ and Y ³ represents H, -(CH ₂ ) _0-3 -CH(NHCOCH ₃ )Z′, -(CH ₂ ) _0-3 -CH(NH ₂ )Z′ or -(CH ₂ ) _0-3 Z′, where Z′ represents H, SO ₃ H, NH ₂ or COOH (wherein “alkyl” preferably represents C _1-10 -alkyl);

R ⁵ represents –L-#1, H, F, NH ₂ , NO ₂ , halogen, SH or -(CH ₂ ) _0-3 Z, wherein Z represents -H, OY ³ , -SY ³ , halogen, NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH;

wherein one of the substituents R ¹ , R ² , R ³ , R ⁴ and R ⁵ represents -L-#1,

L represents a linker and #1 represents a bond to the conjugate or a derivative thereof,

^R6 and ^R7 independently of one another represent H, cyano, (optionally fluorinated) _C1-10 -alkyl, (optionally fluorinated) _C2-10 -alkenyl, (optionally fluorinated) _C2-10 -alkynyl, hydroxy or halogen,

R ⁸ represents (optionally fluorinated) C _1-10 -alkyl, (optionally fluorinated) C _4-10 -cycloalkyl or optionally substituted oxetane; and

R ⁹ represents H, F, CH ₃ , CF ₃ , CH ₂ F or CHF ₂ .

15. The conjugate according to item 14,

wherein R ¹ represents –L-#1, H, -(CH ₂ ) _0-3 Z, wherein Z represents –H, -CO-NY ¹ Y ² , NHY ³ , OY ³ , -SY ³ or –CO-OY ³ ,

wherein Y ¹ and Y ² independently of one another represent H, NH ₂ , -(CH ₂ CH ₂ O) _0-3 -(CH ₂ ) _0-3 Z' or -CH(CH ₂ W)Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, NH ₂ , COOH, -NH-CO-CH ₂ -CH ₂ -CH(NH ₂ )COOH or -(CO-NH-CHY ⁴ ) _1-3 COOH, wherein W represents H or OH, wherein Y ⁴ independently of one another represent linear or branched C _1-6 -alkyl optionally substituted by -NHCONH ₂ , or represent aryl or benzyl optionally substituted by -NH ₂ ;

R ² and R ⁴ independently represent –L-#1, –CO-CHY ⁴ -NHY ⁵ or H, or R ² and R ⁴ together (forming a pyrrolidine ring) represent –CH ₂ -CHR ¹⁰ -, wherein R ¹⁰ represents H, –L-#1, NH ₂ , COOH, SH, OH or SO ₃ H,

wherein Y ⁴ independently of one another represents linear or branched C _1-6 alkyl optionally substituted by -NHCONH ₂ , or represents aryl or benzyl optionally substituted by -NH ₂ , and Y ⁵ represents H or -CO-CHY ⁶ -NH ₂ , wherein Y ⁶ represents linear or branched C _1-6 -alkyl;

A stands for CO,

R ³ represents –(CH ₂ )OH or –L-#1, and

R ⁵ represents –L-#1 or H,

wherein one of the substituents R ¹ , R ² , R ³ , R ⁴ and R ⁵ represents —L-#1.

16. The conjugate according to item 14 or 15, wherein R ⁶ and R ⁷ independently of one another represent H, C _1-3 -alkyl or halogen.

17. The conjugate according to one or more of items 14 to 16, wherein R ⁸ represents C _1-4 -alkyl (preferably tert-butyl).

18. The conjugate according to one or more of items 14 to 17, wherein R ⁹ represents H.

19. The conjugate according to one or more of items 14 to 18, wherein R ⁶ and R ⁷ represent F.

20. The conjugate according to one or more of the preceding items, wherein the linker -L- has one of the following basic structures (i) to (iv):

(i) -(CO) _m –SG1-L1-L2-

(ii) -(CO) _m –L1-SG-L1-L2-

(iii) -(CO) _m –L1-L2-

(iv) –(CO) _m –L1-SG-L2

wherein m is 0 or 1, SG and SG1 are in vivo cleavable groups, L1 independently represent an organic group that is not cleavable in vivo, and L2 represents a coupling group to the conjugate.

21. The conjugate according to item 20, wherein the in vivo cleavable group SG is a 2-8 oligopeptide group, preferably a dipeptide group or a disulfide, hydrazone, acetal or aminal group, and SG1 is a 2-8 oligopeptide group, preferably a dipeptide group.

22. The conjugate according to one or more of items 2 to 21, wherein the linker is attached to a cysteine side chain or a cysteine residue and has the following formula:

§-(CO)m-L1-L2-§§

in

m is 0 or 1;

§ represents the bond to the active substance molecule and

§§ represents a bond to the binding partner peptide or protein, and

-L2-Representative

in

# ¹ refers to the connection point with the sulfur atom of the bond.

# ² refers to the connection point with the group L ¹ ,

L1 represents –(NR ¹⁰ )n-(G1)o-G2-,

wherein R ¹⁰ represents H, NH ₂ or C ₁ -C ₃ -alkyl;

G1 stands for –NHCO- or;

n is 0 or 1;

o is 0 or 1; and

G2 represents a linear or branched hydrocarbon chain having 1 to 100 carbon atoms, which is derived from arylene and/or linear and/or branched and/or cyclic alkylene and may be interrupted one or more times by one or more of the radicals -O-, -S-, -SO-, SO ₂ , -NH-, -CO-, -NMe-, -NHNH-, -SO ₂ NHNH-, -NHCO-, -CONH-, -CONHNH- and a 5- to 10-membered aromatic or non-aromatic heterocycle (preferably) having up to 4 heteroatoms selected from N, O and S, -SO- or -SO ₂ -, where side chains, if present, may be substituted by -NHCONH ₂ , -COOH, -OH, -NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid,

Or represents one of the following groups:

wherein Rx represents H, C ₁ -C ₃ -alkyl or phenyl.

23. The conjugate according to item 22, wherein the hydrocarbon chain is interrupted by one of the following groups:

wherein X represents H or C _1-10 -alkyl which may be optionally substituted by —NHCONH ₂ , —COOH, —OH, NH ₂ , —NH-CNNH ₂ , sulfone, sulfoxide or sulfonic acid.

24. The conjugate according to item 22, wherein the linker has the following formula:

in

#3 represents the bond to the active substance molecule,

#4 represents the bond to the binding peptide or protein,

R ¹¹ represents H or NH ₂ ;

B represents –[(CH ₂ ) _x -(X ⁴ ) _y ] _w -(CH ₂ ) _z -,

w = 0 to 20;

x = 0 to 5;

x = 0 to 5;

y = 0 or 1;

z = 0 to 5; and

X ⁴ represents –O-, –CONH- or –NHCO-.

25. The conjugate according to item 24, wherein R ¹ or R ⁴ represents –L-#1.

26. The conjugate according to one or more of items 21 to 25, wherein the conjugate has one of the following formulae:

in

X ₁ , X ₂ and X ₃ have the same meanings as in item 14,

AK ₁ represents a binder peptide or protein linked via a sulfur atom of the binder; n represents a value from 1 to 20; and L1 represents an optionally branched hydrocarbon radical having 1 to 70 carbon atoms, which represents a linear or branched hydrocarbon chain having 1 to 100 carbon atoms from the group of arylene and/or linear and/or branched and/or cyclic alkylene and may be interrupted one or more times by one or more of the radicals -O-, -S-, -SO-, SO ₂ , -NH-, -CO-, -CONH-, -NHCO-, -NMe-, -NHNH-, -SO ₂ NHNH-, -CONHNH- and a 5- to 10-membered aromatic or non-aromatic heterocycle (preferably) having up to 4 heteroatoms selected from N, O and S or -SO- or -SO ₂ -, where side chains, if present, may be substituted by -NHCONH ₂ , -COOH, -OH, -NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid,

and SG1 is a 2-8 oligopeptide, preferably a dipeptide;

L4 is a single bond or a group -(CO) _y -G4-, where y represents 0 or 1 and G4 represents a linear or branched hydrocarbon chain having 1 to 100 carbon atoms, which is derived from arylene and/or linear and/or branched and/or cyclic alkylene and may be interrupted one or more times by one or more of the groups -O-, -S-, -SO-, SO2, _-NH- , -CO-, -NHCO-, -CONH-, _-NMe- , -NHNH-, -SO2NHNH-, -CONHNH- and a 5- to 10-membered aromatic or non-aromatic heterocycle (preferably) having up to 4 heteroatoms selected from N, O and S, -SO- or _-SO2- , where side chains, if present, may be substituted by _-NHCONH2 , -COOH, -OH, _-NH2 , NH- _CNNH2 , sulfonamide, sulfone, sulfoxide or sulfonic acid.

27. The conjugate according to one or more of items 1 to 21, wherein the linker -L- is attached to a cysteine side chain or a cysteine residue and has the following formula:

in

§ represents the bond to the active substance molecule and

§§ represents the bond to the binding peptide or protein,

m is 0, 1, 2, or 3;

n is 0, 1, or 2;

p is 0 to 20; and

L3 Representative

;

in

o is 0 or 1;

and

G3 represents a linear or branched hydrocarbon chain having 1 to 100 carbon atoms, which is derived from arylene and/or linear and/or branched and/or cyclic alkylene and may be interrupted one or more times by one or more of the radicals -O-, -S-, -SO-, SO ₂ , -NH-, -CO-, -NHCO-, -CONH- and a 5- to 10-membered aromatic or nonaromatic heterocycle (preferably) having up to 4 heteroatoms selected from N, O and S, -NMe-, -NHNH-, -SO ₂ NHNH-, -CONHNH-, -SO- or -SO ₂ -, where side chains, if present, may be substituted by -NHCONH ₂ , -COOH, -OH, sulfones, sulfoxides or sulfonic acids.

28. The conjugate according to item 27, wherein the linker -L- is attached to a cysteine side chain or a cysteine residue and has the following formula:

in

§ represents the bond to the active substance molecule and

§§ represents the bond to the binding peptide or protein,

m is 1;

p is 0;

n is 0;

And L3 represents

;

in

o is 0 or 1; and

G3 represents –(CH ₂ CH ₂ O) _s (CH ₂ ) _t (CONH) _u (CH ₂ CH ₂ O) _v (CH ₂ ) _w -, where

s, t, v and w are each independently 0 to 20 and u is 0 or 1.

29. The conjugate according to item 27 or 28, wherein R ² or R ³ represents –L-#1.

30. The conjugate according to item 29, wherein the conjugate has one of the following formulae:

in

X ₁ , X ₂ and X ₃ have the same meanings as in item 14,

AK ₁ represents a binder peptide or protein linked via a sulfur atom of the binder; n represents a value from 1 to 20; and L2 and L3 represent an optionally branched, linear or branched hydrocarbon chain having 1 to 100 carbon atoms, which is derived from arylene and/or linear and/or branched and/or cyclic alkylene and may be interrupted one or more times by one or more of the groups -O-, -S-, -SO-, SO ₂ , -NH-, -CO-, -NMe-, -NHNH-, -SO ₂ NHNH-, -NHCO-, -CONH-, -CONHNH- and a 5- to 10-membered aromatic or non-aromatic heterocycle (preferably) having up to 4 heteroatoms selected from N, O and S, -SO- or -SO ₂ -, where side chains, if present, may be substituted by -NHCONH ₂ , -COOH, -OH, -NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid.

31. The conjugate according to one or more of items 1 to 21, wherein the linker -L- is attached to the lysine side chain and has the following formula:

-§-(SG) _x -L4-CO-§§

in

§ represents the bond to the active substance molecule and

§§ represents the bond to the binding peptide or protein,

x represents 0 or 1,

SG represents a cleavable group, preferably a 2-8 oligopeptide, particularly preferably a dipeptide,

and

L4 represents a single bond or a group -(CO) _y -G4-, wherein y represents 0 or 1, and

G4 represents a linear or branched hydrocarbon chain having 1 to 100 carbon atoms, which is derived from arylene and/or linear and/or branched and/or cyclic alkylene and may be interrupted one or more times by one or more of the radicals -O-, -S-, -SO-, SO ₂ , -NH-, -CO-, -NHCO-, -CONH-, -NMe-, -NHNH-, -SO ₂ NHNH-, -CONHNH- and a 5- to 10-membered aromatic or nonaromatic heterocycle (preferably) having up to 4 heteroatoms selected from N, O and S or -SO-, where side chains, if present, may be substituted by -NHCONH ₂ , -COOH, -OH, -NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid.

32. The conjugate of a binding peptide or protein according to item 31, wherein the conjugate has one of the following formulae:

in

X ₁ , X ₂ and X ₃ have the same meanings as in item 14,

AK ₂ represents a binder peptide or protein linked via a sulfur atom of the binder; n represents a value from 1 to 20; and L4 represents an optionally linear or branched hydrocarbon chain having 1 to 100 carbon atoms, which is derived from arylene and/or linear and/or branched and/or cyclic alkylene and may be interrupted one or more times by one or more of the groups -O-, -S-, -SO-, SO ₂ , -NH-, -CO-, -NMe-, -NHNH-, -SO ₂ NHNH-, -NHCO-, -CONH-, -CONHNH- and a 5- to 10-membered aromatic or non-aromatic heterocycle (preferably) having up to 4 heteroatoms selected from N, O and S, -SO- or -SO ₂ -, where side chains, if present, may be substituted by -NHCONH ₂ , -COOH, -OH, -NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid, and

SG1 represents a cleavable group, preferably a 2-8 oligopeptide, particularly preferably a dipeptide.

33. The conjugate according to one or more of items 10 to 32, wherein the anti-TWEAKR antibody is an agonistic antibody.

34. The conjugate according to one or more of items 10 to 33, comprising:

A variable heavy chain comprising:

a. A heavy chain CDR1 encoded by an amino acid sequence comprising the formula PYPMX (SEQ ID NO: 171), wherein X is I or M;

b. CDR2 of the heavy chain comprising an amino acid sequence of the formula YISPSGGXTHYADSVKG (SEQ ID NO: 172), wherein X is S or K; and

c. a heavy chain CDR3 encoded by an amino acid sequence comprising the formula GGDTYFDYFDY (SEQ ID NO: 173);

and a variable light chain comprising:

d. a light chain CDR1 encoded by an amino acid sequence comprising the formula RASQSISXYLN (SEQ ID NO: 174), wherein X is G or S;

e. CDR2 of the light chain encoded by an amino acid sequence comprising the formula XASSLQS (SEQ ID NO: 175), wherein X is Q, A or N; and

f. A CDR3 of the light chain encoded by an amino acid sequence comprising the formula QQSYXXPXIT (SEQ ID NO: 176), wherein X at position 5 is T or S, X at position 6 is T or S, and X at position 8 is G or F.

35. The conjugate according to one or more of items 10 to 34, comprising:

a. a variable sequence of the heavy chain as shown in SEQ ID NO: 10 and a variable sequence of the light chain as shown in SEQ ID NO: 9, or

b. the variable sequence of the heavy chain as shown in SEQ ID NO: 20 and the variable sequence of the light chain as shown in SEQ ID NO: 19, or

c. the variable sequence of the heavy chain as shown in SEQ ID NO: 30 and the variable sequence of the light chain as shown in SEQ ID NO: 29, or

d. a heavy chain variable sequence as shown in SEQ ID NO: 40 and a light chain variable sequence as shown in SEQ ID NO: 39, or

e. the variable sequence of the heavy chain as shown in SEQ ID NO: 50 and the variable sequence of the light chain as shown in SEQ ID NO: 49, or

f. the variable sequence of the heavy chain as shown in SEQ ID NO: 60 and the variable sequence of the light chain as shown in SEQ ID NO: 59, or

g. the variable sequence of the heavy chain as shown in SEQ ID NO: 70 and the variable sequence of the light chain as shown in SEQ ID NO: 69, or

h. the variable sequence of the heavy chain as shown in SEQ ID NO: 80 and the variable sequence of the light chain as shown in SEQ ID NO: 79, or

i. the variable sequence of the heavy chain as shown in SEQ ID NO: 90 and the variable sequence of the light chain as shown in SEQ ID NO: 89, or

j. the variable sequence of the heavy chain as shown in SEQ ID NO: 100 and the variable sequence of the light chain as shown in SEQ ID NO: 99, or

k. the variable sequence of the heavy chain as shown in SEQ ID NO: 110 and the variable sequence of the light chain as shown in SEQ ID NO: 109, or

1. The variable sequence of the heavy chain is shown in SEQ ID NO: 120 and the variable sequence of the light chain is shown in SEQ ID NO: 119.

36. The conjugate according to one or more of items 10 to 35, wherein the antibody is an IgG antibody.

37. The conjugate according to one or more of items 10 to 36, comprising:

a. The heavy chain sequence as shown in SEQ ID NO: 2 and the light chain sequence as shown in SEQ ID NO: 1, or

b. the sequence of the heavy chain as shown in SEQ ID NO: 12 and the sequence of the light chain as shown in SEQ ID NO: 11, or

c. the heavy chain sequence as shown in SEQ ID NO: 22 and the light chain sequence as shown in SEQ ID NO: 21, or

d. the heavy chain sequence as shown in SEQ ID NO: 32 and the light chain sequence as shown in SEQ ID NO: 31, or

e. the heavy chain sequence as shown in SEQ ID NO: 42 and the light chain sequence as shown in SEQ ID NO: 41, or

f. the heavy chain sequence as shown in SEQ ID NO: 52 and the light chain sequence as shown in SEQ ID NO: 51, or

g. the heavy chain sequence as shown in SEQ ID NO: 62 and the light chain sequence as shown in SEQ ID NO: 61, or

h. the sequence of the heavy chain as shown in SEQ ID NO: 72 and the sequence of the light chain as shown in SEQ ID NO: 71, or

i. the sequence of the heavy chain as shown in SEQ ID NO: 82 and the sequence of the light chain as shown in SEQ ID NO: 81, or

j. the sequence of the heavy chain as shown in SEQ ID NO: 92 and the sequence of the light chain as shown in SEQ ID NO: 91, or

k. the sequence of the heavy chain as shown in SEQ ID NO: 102 and the sequence of the light chain as shown in SEQ ID NO: 101, or

1. The sequence of the heavy chain is shown in SEQ ID NO: 112 and the sequence of the light chain is shown in SEQ ID NO: 111.

38. Spindle conjugate according to one or more of the preceding items, wherein the conjugate has 1 to 10, preferably 2 to 8, molecules of active substance per binding partner peptide or protein.

39. A process for preparing a conjugate according to item 26 or 30, wherein a compound of one of the following formulae, preferably in the form of its trifluoroacetate salt, is linked to a cysteine residue of an optionally previously partially reduced binder peptide or protein, wherein the compound is preferably used in a 2- to 12-fold molar excess relative to the binder peptide or protein:

Where R represents -H or –COOH,

wherein K represents a linear or branched chain optionally substituted by C ₁ -C ₆ -alkoxy or -OH, C ₁ -C ₆ -alkyl, and

wherein X ₁ , X ₂ , X ₃ , SG1, L1, L2, L3 and L4 have the same meanings as in item 26 or 30.

40. A method for preparing a conjugate according to item 32, wherein a compound of one of the following formulae, preferably in the form of its trifluoroacetate salt, is linked to a lysine residue of a binding peptide or protein, wherein the compound is preferably used in a 2- to 12-fold molar excess relative to the binding peptide or protein:

wherein X ₁ , X ₂ , X ₃ , SG1 and L4 have the same meanings as in item 32.

41. A compound of one of the following formulae:

Where R represents -H or –COOH,

wherein K represents a linear or branched chain optionally substituted by C ₁ -C ₆ -alkoxy or -OH, C ₁ -C ₆ -alkyl, and

wherein X ₁ , X ₂ , X ₃ , SG1, L1, L2, L3 and L4 have the same meanings as in item 26, 30 or 32.

42. Compounds of general formula (III) and their salts, solvates and salts of solvates:

in

_X1 represents N, _X2 represents N and _X3 represents C, or _X1 represents CH, _X2 represents C and _X3 represents N, or _X1 represents NH, _X2 represents C and _X3 represents C, or _X1 represents CH, _X2 represents N and _X3 represents C;

R ¹ represents –L-BINDER, H or -(CH ₂ ) _0-3 Z, wherein Z represents -H, -NHY ³ , -OY ³ , -SY ³ , halogen, -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ , -(CH ₂ CH ₂ O) _0-3 -(CH ₂ ) _0-3 Z' or -CH(CH ₂ W)Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, NH ₂ , SO ₃ H, COOH, -NH-CO-CH ₂ -CH ₂ -CH(NH ₂ )COOH or -(CO-NH-CHY ⁴ ) _1-3 COOH; wherein W represents H or OH;

wherein Y ⁴ independently of one another represent linear or branched C _1-6 -alkyl optionally substituted by -NHCONH ₂ , or represent aryl or benzyl optionally substituted by -NH ₂ ;

R ² and R ⁴ independently represent -L-BINDER, H, -CO-CHY ⁴ -NHY ⁵ or -(CH ₂ ) _0-3 Z, or R ² and R ⁴ together (forming a pyrrolidine ring) represent -CH ₂ -CHR ¹⁰ - or -CHR ¹⁰ -CH ₂ -, wherein R ¹⁰ represents L-#1, H, NH ₂ , SO ₃ H, COOH, SH or OH,

wherein Z represents -H, halogen, -OY ³ , -SY ³ , NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH;

wherein Y ⁴ independently of one another represents linear or branched C _1-6 alkyl optionally substituted by -NHCONH ₂ , or represents aryl or benzyl optionally substituted by -NH ₂ , and Y ⁵ represents H or -CO-CHY ⁶ -NH ₂ , wherein Y ⁶ represents linear or branched C _1-6 -alkyl;

A represents CO, SO, SO ₂ , SO ₂ NH or CNNH;

R ³ represents –L-BINDER or optionally substituted alkyl, aryl, heteroaryl, heteroalkyl, heterocycloalkyl, preferably –L-#1 or C _1-10 -alkyl, C _6-10 -aryl or C _{6-10 -arylalkyl, C 5-10} -heteroalkyl, C _1-10 -alkyl-OC _6-10 -aryl or C _5-10 -heterocycloalkyl, which may be substituted by 1-3 –OH groups, 1-3 halogen atoms, _1-3 haloalkyl groups (each having 1-3 halogen atoms), 1-3 O-alkyl groups, 1-3 –SH groups, 1-3 –S-alkyl groups, 1-3 –O-CO-alkyl groups, 1-3 –O-CO-NH-alkyl groups, 1-3 –NH-CO-alkyl groups, 1-3 –NH-CO-NH-alkyl groups, 1-3 –S(O) _n -alkyl groups, 1-3 –SO ₂ -NH-alkyl, 1-3 -NH-alkyl, 1-3 -N(alkyl) ₂ groups, 1-3 -NH ₂ groups or 1-3 -(CH ₂ ) _0-3 Z groups, where Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ , where Y ¹ and Y ² independently of one another represent H, NH ₂ or -(CH ₂ ) _0-3 Z ', and Y ³ represents H, -(CH ₂ ) _0-3 -CH(NHCOCH ₃ )Z ', -(CH ₂ ) _0-3 -CH(NH ₂ )Z ' or -(CH ₂ ) _0-3 Z ', where Z ' represents H, SO ₃ H, NH ₂ or COOH (wherein "alkyl" preferably represents C _1-10 -alkyl);

R ⁵ represents -L-BINDER, H, F, NH ₂ , NO ₂ , halogen, SH or -(CH ₂ ) _0-3 Z, wherein Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH;

wherein L represents a linker and BINDER represents a binder or a derivative thereof, wherein the binder can optionally be linked to a plurality of active substance molecules,

^R6 and ^R7 independently of one another represent H, cyano, (optionally fluorinated) _C1-10 -alkyl, (optionally fluorinated) _C2-10 -alkenyl, (optionally fluorinated) _C2-10 -alkynyl, hydroxy or halogen,

R ⁸ represents (optionally fluorinated) C _1-10 -alkyl, (optionally fluorinated) C _4-10 -cycloalkyl or optionally substituted oxetane; and

R ⁹ represents H, F, CH ₃ , CF ₃ , CH ₂ F or CHF ₂ .

43. Compounds of general formula (IV) and their salts, solvates and salts of solvates:

in

_X1 represents N, _X2 represents C and _X3 represents N;

R ¹ represents –L-BINDER, H or -(CH ₂ ) _0-3 Z, wherein Z represents -H, -NHY ³ , -OY ³ , -SY ³ , halogen, -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ , -(CH ₂ CH ₂ O) _0-3 -(CH ₂ ) _0-3 Z' or -CH(CH ₂ W)Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, NH ₂ , SO ₃ H, COOH, -NH-CO-CH ₂ -CH ₂ -CH(NH ₂ )COOH or -(CO-NH-CHY ⁴ ) _1-3 COOH; wherein W represents H or OH;

wherein Y ⁴ independently of one another represent a linear or branched C _1-6 -alkyl group optionally substituted by -NHCONH ₂ , or represent an aryl group or a benzyl group optionally substituted by -NH ₂ ;

R ² and R ⁴ independently represent -L-BINDER, H, -CO-CHY ⁴ -NHY ⁵ or -(CH ₂ ) _0-3 Z, or R ² and R ⁴ together (forming a pyrrolidine ring) represent -CH ₂ -CHR ¹⁰ - or -CHR ¹⁰ -CH ₂ -, wherein R ¹⁰ represents L-#1, H, NH ₂ , SO ₃ H, COOH, SH or OH,

wherein Z represents -H, halogen, -OY ³ , -SY ³ , NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH;

wherein Y ⁴ independently of one another represents linear or branched C _1-6 alkyl optionally substituted by -NHCONH ₂ or represents aryl or benzyl optionally substituted by -NH ₂ , and Y ⁵ represents H or -CO-CHY ⁶ -NH ₂ , wherein Y ⁶ represents linear or branched C _1-6 -alkyl;

A represents CO, SO, SO ₂ , SO ₂ NH or CNNH;

R ³ represents –L-BINDER or optionally substituted alkyl, aryl, heteroaryl, heteroalkyl or heterocycloalkyl, preferably –L-#1 or C _1-10 -alkyl, C _6-10 -aryl, C 6-10 -aralkyl, C _5-10 -heteroalkyl, C _1-10 -alkyl-OC _6-10 -aryl- or C _5-10 -heterocycloalkyl, which may be substituted by 1-3 –OH groups, 1-3 halogen atoms, _1-3 haloalkyl groups (each having 1-3 halogen atoms), 1-3 O-alkyl groups, 1-3 –SH groups, 1-3 –S-alkyl groups, 1-3 –O-CO-alkyl groups, 1-3 –O-CO-NH-alkyl groups, 1-3 –NH-CO-alkyl groups, 1-3 –NH-CO-NH-alkyl groups, 1-3 –S(O) _n -alkyl groups, 1-3 –SO ₂ -NH-alkyl, 1-3 -NH-alkyl, 1-3 -N(alkyl) ₂ groups, 1-3 -NH ₂ groups or 1-3 -(CH ₂ ) _0-3 Z groups, where Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ , where Y ¹ and Y ² independently of one another represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H, -(CH ₂ ) _0-3 -CH(NHCOCH ₃ )Z', -(CH ₂ ) _0-3 -CH(NH ₂ )Z' or -(CH ₂ ) _0-3 Z', where Z' represents H, SO ₃ H, NH ₂ or COOH (wherein "alkyl" preferably represents C _1-10 -alkyl);

R ⁵ represents -L-BINDER, H, F, NH ₂ , NO ₂ , halogen, SH or -(CH ₂ ) _0-3 Z, wherein Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ ,

wherein Y ¹ and Y ² independently represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH;

wherein L represents a linker and BINDER represents a binder or a derivative thereof, wherein the binder can optionally be linked to a plurality of active substance molecules,

^R6 and ^R7 independently of one another represent H, cyano, (optionally fluorinated) _C1-10 -alkyl, (optionally fluorinated) _C2-10 -alkenyl, (optionally fluorinated) _C2-10 -alkynyl, hydroxy or halogen,

R ⁸ represents (optionally fluorinated) C _1-10 -alkyl, (optionally fluorinated) C _4-10 -cycloalkyl or optionally substituted oxetane; and

R ⁹ represents H, F, CH ₃ , CF ₃ , CH ₂ F or CHF ₂ ;

Provided that R ¹ , R ² and R ⁴ do not represent H at the same time.

44. The compound according to item 43, wherein Z represents Cl or Br.

45. The compound according to item 43, wherein R ¹ represents -(CH ₂ ) _0-3 Z, wherein Z represents -CO-NY ¹ Y ² , wherein Y ² represents -(CH ₂ CH ₂ O) _0-3 -(CH ₂ ) _0-3 Z', and Y ¹ represents H, NH ₂ or -(CH ₂ CH ₂ O) _0-3 -(CH ₂ ) _0-3 Z'.

46. Compounds according to one or more of items 43-44, wherein Y ¹ represents H, Y ² represents —(CH ₂ CH ₂ O) ₃ —CH ₂ CH ₂ Z′ and Z′ represents —COOH.

47. Compounds according to one or more of items 43-44, wherein Y ¹ represents H, Y ² represents —CH ₂ CH ₂ Z′ and Z′ represents —(CONHCHY ⁴ ) ₂ COOH.

48. The compound according to item 47, wherein Y ⁴ represents a linear or branched C _1-6 -alkyl group optionally substituted by —NHCONH ₂ .

49. The compound according to item 48, wherein at least one representative of Y ⁴ is selected from isopropyl and -CH ₃ .

50. Compounds according to one or more of items 43 and 45, wherein Y ¹ represents H, Y ² represents -CH ₂ CH ₂ Z', Z' represents -CONHCHY ⁴ COOH and Y ⁴ represents aryl or benzyl optionally substituted by -NH ₂ .

51. The compound according to item 50, wherein Y ⁴ represents aminobenzyl.

52. The compound according to item 43, wherein R ² represents —(CH ₂ ) _0-3 Z and Z represents —SY ³ .

53. The compound according to item 43, wherein R ⁴ represents -CO-CHY ⁴ -NHY ⁵ and Y ⁵ represents H.

54. The compound according to item 43, wherein R ⁴ represents —CO-CHY ⁴ —NHY ⁵ and Y ⁵ represents —CO-CHY ⁶ —NH ₂ .

55. Compounds according to one or more of items 53 and 54, wherein Y ⁴ represents linear or branched C _1-6 -alkyl optionally substituted by —NHCONH ₂ .

56. The compound according to one or more of items 43 to 55, wherein the compound has one of the following formulae:

(15S,19R)-15-amino-19-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-18-glycoloyl-20,20-dimethyl-14-oxo-4,7,10-trioxa-13,18-diazaheneicosane-1-oic acid;

N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl-L-valyl-N ⁵ -carbamoyl-L-ornithine;

N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl-L-alanyl-N ⁵ -carbamoyl-L-ornithine;

N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl-4-amino-L-phenylalanine;

N-{(1R)-1-[1-(3-aminobenzyl)-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-N-(3-aminopropyl)-2-hydroxyacetamide (1:1);

(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butyric acid;

N-[(3S)-3-amino-4-hydrazino-4-oxobutyl]-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide (1:1);

N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-N-[(3S)-3,4-diaminobutyl]-2-hydroxyacetamide (1:1);

N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butyryl}-β-alanine;

(2S)-2-amino-N-(2-aminoethyl)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butanamide (2:1);

(1-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butanoyl}hydrazino)acetic acid;

N-[3-Amino-2-(sulfanylmethyl)propyl]-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide hydrochloride (1:1);

4-amino-N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}benzamide (2:1);

N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-N5-carbamoyl-L-ornithinamide (1:1);

L-valyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-N5-carbamoyl-L-ornithinamide (1:1);

L-Valyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-N5-carbamoyl-L-ornithinamide (1:1).

57. A pharmaceutical composition comprising a conjugate according to one or more of items 1 to 38 or a compound according to items 42 to 56 and an inert, non-toxic, pharmaceutically acceptable auxiliary.

58. The conjugate according to one or more of items 1 to 38 or the compound according to items 42 to 56 for use in a method for treating and/or preventing a disease.

59. A conjugate according to one or more of items 1 to 38 or a compound according to items 42 to 56 for use in a method of treating a hyperproliferative and/or angiogenic disease.

60. The conjugate according to one or more of items 1 to 38 or the compound according to items 42 to 56, wherein R ^3a or R ³ represents -L-BINDER or substituted alkyl, aryl or heteroaryl, preferably -L-#1 or C _1-10 -alkyl, C _6-10 -aryl or C _6-10 -aralkyl or C _5-10 -heteroalkyl, which may be substituted by -OH, O-alkyl, SH, S-alkyl, O-CO-alkyl, O-CO-NH-alkyl, NH-CO-alkyl, NH-CO-NH-alkyl, S(O) _n -alkyl, SO ₂ -NH-alkyl, NH-alkyl, N(alkyl) ₂ , NH ₂ or -(CH ₂ ) _0-3 Z, wherein Z represents -H, halogen, -OY ³ , -SY ³ , -NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ , wherein Y ^{Y 1} and Y ² independently of one another represent H, NH ₂ or -(CH ₂ ) _0-3 Z', and Y ³ represents H or -(CH ₂ ) _0-3 Z', wherein Z' represents H, SO ₃ H, NH ₂ or COOH (wherein "alkyl" preferably represents C _1-10 -alkyl).

61. Method for treating and/or preventing hyperproliferative and/or angiogenic diseases in humans and animals using an effective amount of at least one conjugate according to one or more of items 1 to 38 or a compound according to items 42 to 56.

Claims (91)

1. A conjugate of a conjugate or its derivative with one or more active substance molecules, wherein the active substance molecule is a spindle kinesin inhibitor linked to the conjugate via a linker L, wherein the spindle kinesin inhibitor has the following formula (IIa) and its salt: in _X1 represents N, _X2 represents N, and _X3 represents C; or _X1 represents CH or CF, _X2 represents C and _X3 represents N; ^R1 represents H, -L-#1, -MOD, or -( _CH2 ) _0-3Z , where Z represents -H, ^-NHY3 , ^-OY3 , ^-SY3 , halogen, -CO- ^NY1Y2 , or -CO- ^{OY3 . Y1} and ^Y2 independently represent H, _NH2 , -( _CH2CH2O ) _O-3- ( _CH2 )O- _3Z ′, -( _CH2 ) _O-3Z ′, or -CH( _CH2W )Z′, and ^Y3 represents H or -( _CH2 ) _{O -3Z} ′, where Z′ represents H, _NH2 , _SO2H , COOH, -NH-CO- _CH2 - _CH2 -CH( _NH2 )COOH, or -(CO-NH- ^CHY4 ) _1-3COOH , where W represents H or OH. ^Y4 independently represents either a straight-chain or branched _C1-6 -alkyl group optionally substituted with -NHCONH ₂ , or an aryl or benzyl group optionally substituted with -NH ₂ ; ^R2 represents -L-#1, H, -MOD, -CO-CHY4 ^{-NHY5 or} -( _CH2 ) _0-3Z , or ^R2 and ^R4 together form a pyrrolidine ring representing _-CH2- ^CHR10- or ^-CHR10 _-CH2- , where ^R10 represents L-#1, H, _NH2 , _SO3H , COOH, SH or OH; Where Z represents -H, halogen, ^-OY3 , ^-SY3 , ^NHY3 , -CO- ^NY1Y2 , or -CO- ^{OY3 . Y1} and ^Y2 independently represent H, _NH2 or -( _CH2 ) _O- 3Z′, and ^Y3 represents H or -( _CH2 ) _O- 3Z′, where Z′ represents H, _SO3H , _NH2 or COOH; ^Y4 independently represents a straight-chain or branched _C1-6 alkyl group optionally substituted with -NHCONH ₂ , or an aryl or benzyl group optionally substituted with -NH ₂ , and ^Y5 represents H or -CO-CHY ⁶ -NH ₂ , wherein ^Y6 represents a straight-chain or branched _C1-6 alkyl group. R ⁴ represents -L-#1, H, -CO-CHY ⁴ -NHY ⁵ , or -(CH ₂ ) _0-3 Z, Where Z represents -H, halogen, ^-OY3 , ^-SY3 , ^NHY3 , -CO- ^NY1Y2 , or -CO- ^{OY3 . Y1} and ^Y2 independently represent H, _NH2 or -( _CH2 ) _O- 3Z′, and ^Y3 represents H or -( _CH2 ) _O- 3Z′, where Z′ represents H, _SO3H , _NH2 or COOH; ^Y4 independently represents a straight-chain or branched _C1-6 alkyl group optionally substituted with -NHCONH ₂ , or an aryl or benzyl group optionally substituted with -NH ₂ , and ^Y5 represents H or -CO-CHY ⁶ -NH ₂ , wherein ^Y6 represents a straight-chain or branched _C1-6 alkyl group. ^R2 and ^R4 together form a pyrrolidine ring representing _-CH2- ^CHR10- or ^-CHR10 - _CH2- , where ^R10 represents L-#1, H, _NH2 , _SO3H , COOH, SH, or OH; A represents CO, SO, _SO₂ , _SO₂NH , or CNNH; R ³ represents -L-#1, -MOD, or optionally substituted alkyl, cycloalkyl, aryl, heteroaryl, heteroalkyl, or heterocycloalkyl. R ⁵ represents -L-#1, H, halogen; ^R6 and ^R7 independently represent H, optionally fluorinated _C1-10 alkyl, or halogen. R ⁸ represents a _C1-10 alkyl group that is optionally fluorinated; One of the substituents ^R1 , ^R2 , ^R3 , ^R4 , ^R5 , and ^R10 represents -L-#1. L represents the linker and #1 represents the bond with the linker or its derivative. R ⁹ represents H, F, CH ₃ , CF ₃ , CH _2F , or CHF ₂ ; Where -MOD represents -(NR ¹⁰ ) _n -(G1) _o -G2-H, R ¹⁰ represents H or _C1 - _C3 -alkyl; G1 represents -NHCO-, -CONH-, or one of them. If G1 represents -NHCO-, then ^R10 does not represent _NH2 . n is 0 or 1; o is 0 or 1; and G2 represents a straight-chain and/or branched hydrocarbon group having 1 to 10 carbon atoms and being interrupted once or more by one or more of the groups -O-, -S-, -SO-, _SO₂ , ^-NRy- , ^-NRyCO- , ^CONRy- , ^-NRyNRy- , _{-SO₂NRyNRy-} , ^-CONRyNRy- , -CO-, ^-CRx =NO-, wherein if a hydrocarbon chain is present, including a ^side chain, it may be substituted with _-NHCONH₂ , ^-COOH , ^-OH , -NH₂, NH- _CNNH₂ , sulfonamide, sulfone, sulfoxide, or sulfonic acid, wherein the ^conjugate or its derivative is a human, humanized, or chimeric monoclonal antibody or _its antigen-binding fragment. Where R ^_y represents H, phenyl, C<sub> ₁ -C_₁₀-alkyl, C <sub>₂ -C_₁₀-alkenyl, or C<sub> _2- C_₁₀-alkynyl, which may be substituted by -NHCONH_₂, -COOH, -OH, -NH_₂ , NH-CNNH_₂, sulfonamide, sulfone, sulfoxide, or sulfonic acid, and Where ^Rx represents H, _C1 - _C3 -alkyl or phenyl. 2. The coupling according to claim 1, wherein ^R3 represents -L-#1 or _C1-10 -alkyl, _C6-10 -aryl or C6-10 -aralkyl, _C5-10 -heteroalkyl, _C1-10 -alkyl- _OC6-10 -aryl or _C5-10 -heterocyclic alkyl, which may be substituted with 1-3 -OH groups, _1-3 halogen atoms, 1-3 haloalkyl groups each having 1-3 halogen atoms, 1-3 O-alkyl groups, 1-3 -SH groups, 1-3 -S-alkyl groups, 1-3 -O-CO-alkyl groups, 1-3 -O-CO-NH-alkyl groups, 1-3 -NH-CO-alkyl groups, 1-3 -NH-CO-NH-alkyl groups, 1-3 -S(O) _n -alkyl groups, 1-3 -SO2 _- NH-alkyl groups, 1-3 -NH-alkyl groups, 1-3 -N(alkyl) The substituted group consists of ₂ groups, 1-3 _-NH2 groups, or 1-3 -( _CH2 ) _0-3Z groups, where Z represents -H, halogen, ^-OY3 , ^-SY3 , ^-NHY3 , -CO- ^NY1Y2 , or -CO- ^OY3 , where ^{Y1 and Y2} independently represent H, _NH2 , or -( _CH2 ) _0-3Z ′ and ^Y3 represents H, -( _CH2 ) _0-3 -CH( _NHCOCH3 )Z′, -( _CH2 ) _0-3 -CH( _NH2 )Z′, or -( _CH2 ) _0-3Z ′, where Z′ represents H, _SO3H , _NH2 , or COOH, and "alkyl" represents _C1-10 -alkyl, where n is 0 or 1. 3. The conjugate according to claim 1, wherein ^R5 represents F, Cl, or Br. 4. The coupling according to claim 1, wherein ^R6 and ^R7 independently represent F, Cl, and Br. 5. The coupling compound according to claim 1, wherein the group -MOD has at least one group -COOH. 6. The conjugate according to claim 1, wherein _X1 represents CH, _X2 represents C and _X3 represents N. 7. The conjugate according to claim 1 or 6, wherein the substituent ^R1 represents -L-#1. 8. The conjugate according to claim 1 and its salt, wherein the active substance molecule-linker is represented by general formula (II): in _X1 represents N, _X2 represents N, and _X3 represents C; or _X1 represents CH, _X2 represents C, and _X3 represents N; ^R1 represents H, -L-#1, or -( _CH2 ) _0-3Z , where Z represents -H, ^-NHY3 , ^-OY3 , ^-SY3 , halogen, -CO- ^NY1Y2 , or -CO- ^{OY3 . Y1} and ^Y2 independently represent H, _NH2 , -(CH2CH2O) _0-3- ( _CH2 ) _0-3Z ′ or -CH( _CH2W )Z′, and ^Y3 represents H or - _{( CH2} ) _0-3Z ′, where Z′ represents H, _NH2 , SO3H, COOH, -NH _- CO-CH2- _{CH2 - CH} ( _NH2 )COOH or -(CO-NH- ^CHY4 ) _1-3COOH , where W represents H or OH, and ^Y4 independently represent a straight-chain or branched _C1-6 -alkyl optionally substituted with _-NHCONH2 , or an aryl or benzyl optionally substituted with _-NH2 ; ^R2 and ^R4 independently represent -L-#1, H, -CO-CHY4 _- ^NHY5 , or -( _CH2 ) ^0-3Z , or ^R2 and ^R4 together form a pyrrolidine ring representing _-CH2- ^CHR10- or ^-CHR10 - _CH2- , where ^R10 represents L-#1, H, _NH2 , _SO3H , COOH, SH, or OH. Where Z represents -H, OY ³ , -SY ³ , halogen, NHY ³ , -CO-NY ¹ Y ² or -CO-OY ³ , ^Y1 and ^Y2 independently represent H, _NH2 or -( _CH2 ) _O- 3Z′, and ^Y3 represents H or -( _CH2 ) _O- 3Z′, where Z′ represents H, _SO3H , _NH2 or COOH; ^Y4 independently represents a straight-chain or branched _C1-6 alkyl group optionally substituted with -NHCONH ₂ , or an aryl or benzyl group optionally substituted with -NH ₂ , and ^Y5 represents H or -CO-CHY ⁶ -NH ₂ , wherein ^Y6 represents a straight-chain or branched _C1-6 alkyl group. A represents CO, SO, _SO₂ , _SO₂NH , or CNNH; R ³ represents -L-#1 or optionally substituted alkyl, aryl, heteroaryl, heteroalkyl or heterocyclic alkyl; R ⁵ represents -L-#1, H, halogen; One of the substituents ^R1 , ^R2 , ^R3 , ^R4 , and ^R5 represents -L-#1. L represents the linker and #1 represents the bond with the linker or its derivative. ^R6 and ^R7 independently represent H, _C1-3 -alkyl, or halogen. R ⁸ represents _C1-4 -alkyl; and ^R9 represents H, F, _CH3 , _CF3 , _CH2F , or _CHF2 , and the conjugate or its derivative is a human, humanized, or chimeric monoclonal antibody or its antigen-binding fragment. 9. The coupling according to claim 8, wherein ^R3 represents -L-#1 or _C1-10 -alkyl, _{C6-10 -aryl, C6-10} -aralkyl, _C5-10 -heteroalkyl, _C1-10 -alkyl-OC6-10 -aryl- or _C5-10 -heterocyclic alkyl, which may be 1-3 -OH groups, _1-3 halogen atoms, _1-3 haloalkyl groups each having 1-3 halogen atoms, 1-3 O-alkyl, 1-3 -SH groups, 1-3 -S-alkyl, 1-3 -O-CO-alkyl, 1-3 -O-CO-NH-alkyl, 1-3 -NH-CO-alkyl, 1-3 -NH-CO-NH-alkyl, 1-3 -S(O)n-alkyl, 1-3 -SO2 _- NH-alkyl, 1-3 -NH-alkyl, 1-3 -N(alkyl) The substitution is ₂ groups, 1-3 _-NH2 groups or 1-3 -( _CH2 ) _0-3Z groups, where Z represents -H, halogen, ^-OY3 , ^-SY3 , ^-NHY3 , -CO- ^NY1Y2 or -CO- ^OY3 , where ^{Y1 and Y2} independently represent H, _NH2 or -( _CH2 ) _0-3Z ′, and ^Y3 represents H, -( _CH2 ) _0-3 -CH( _NHCOCH3 )Z′, -( _CH2 ) _0-3 -CH( _NH2 )Z′ or -( _CH2 ) _0-3Z ′, where Z′ represents H, _SO3H , _NH2 or COOH, where "alkyl" represents _C1-10 -alkyl, and n is 0 or 1. 10. The coupling according to claim 8, Where ^R1 represents -L-#1, H, -( _CH2 ) _0-3Z , and Z represents -H, -CO- ^NY1Y2 , ^NHY3 , ^OY3 , ^{-SY3 ,} or -CO- ^OY3 . ^Y1 and ^Y2 independently represent H, _NH2 , -( _CH2CH2O ) _0-3- ( _CH2 ) _0-3Z ′ or -CH( _CH2W )Z′, and ^Y3 represents H or -( _CH2 ) _0-3Z ′, where Z′ represents H, _NH2 , COOH, -NH-CO-CH2- _CH2 - _CH ( _{NH2 )} COOH or -(CO-NH- ^CHY4 ) _1-3COOH , where W represents H or OH, and ^Y4 independently represent a straight-chain or branched _C1-6 -alkyl optionally substituted with _-NHCONH2 , or _an aryl or benzyl optionally substituted with _-NH2 ; ^R2 and ^R4 can independently represent -L-#1, -CO-CHY4 ^{-NHY5 ,} or H, or ^R2 and ^R4 together can form a pyrrolidine ring representing _-CH2- ^CHR10- , where ^R10 represents H, -L-#1, _NH2 , COOH, SH, OH, or _SO3H . ^Y4 independently represents a straight-chain or branched _C1-6 alkyl group optionally substituted with -NHCONH ₂ , or an aryl or benzyl group optionally substituted with -NH ₂ , and ^Y5 represents H or -CO-CHY ⁶ -NH ₂ , wherein ^Y6 represents a straight-chain or branched _C1-6 alkyl group. A represents CO. R ³ represents -(CH ₂ )OH or -L-#1, and R ⁵ represents -L-#1 or H. One of the substituents ^R1 , ^R2 , ^R3 , ^R4 , and ^R5 represents -L-#1. 11. The coupling of claim 8, wherein R ⁸ represents tert-butyl. 12. The coupling according to claim 8, wherein R ⁹ represents H. 13. The coupling of claim 8, wherein ^R6 and ^R7 represent F. 14. The conjugate according to claim 1, wherein each active substance molecule is linked via a linker to a different amino acid of the conjugate or a derivative thereof. 15. The conjugate of claim 1, wherein the conjugate has an average of 1.2 to 20 active substance molecules per conjugate. 16. The conjugate of claim 1, wherein the conjugate represents an antibody or a derivative thereof. 17. The conjugate of claim 1, wherein the conjugate is bound to a cancer target molecule. 18. The conjugate of claim 17, wherein the conjugate binds to an extracellular target molecule. 19. The conjugate of claim 18, wherein the conjugate is internalized by the cell expressing the target molecule after binding to the extracellular target molecule and processed intracellularly by means of lysosomes. 20. The conjugate of claim 1, wherein the conjugate is an anti-HER2 antibody, an anti-EGFR antibody, an anti-TWEAKR antibody, or an antigen-binding fragment thereof. 21. The conjugate of claim 20, wherein the anti-TWEAKR antibody specifically binds to amino acid D (D47) at position 47 of TWEAKR (SEQ ID NO: 169). 22. The conjugate of claim 21, wherein the anti-TWEAKR antibody is anti-TWEAKR antibody TPP-2090. 23. The conjugate of claim 20, wherein the conjugate is an anti-EGFR antibody and R3 represents -L-#1. 24. The coupling according to claim 1, wherein the connector-L- has one of the following basic structures (i) to (iv): -(CO) _m -SG1-L1-L2- (ii)-(CO) _m -L1-SG-L1-L2- (iii)-(CO) _m -L1-L2- (iv)-(CO) _m -L1-SG-L2 Where m is 0 or 1, SG and SG1 are cleavable groups in vivo, L1 represents organic groups that are not cleavable in vivo independently, and L2 represents coupling groups with the complex. 25. The conjugate of claim 24, wherein the in vivo cleavable group SG is a 2-8 oligopeptide group or a disulfide, hydrazone, acetal or acetalamine, and SG1 is a 2-8 oligopeptide group. 26. The conjugate of claim 25, wherein the in vivo cleavable group SG is a dipeptide group. 27. The conjugate of claim 25, wherein SG1 is a dipeptide group. 28. The conjugate of claim 1, wherein the linker is attached to a cysteine side chain or a cysteine residue and has the following formula: §-(CO)m-L1-L2-§§ in m is 0 or 1; § represents the bond that connects to the active substance molecule and §§ represents the bond in the linker, and -L2- represents in # ¹ refers to the connection point with the sulfur atom of the bonded organism. # ² refers to the connection point with group ^L1 . L1 represents -(NR ¹⁰ )n-(G1)o-G2-, Where R ¹⁰ represents H, NH ₂ , or C ₁ -C ₃ -alkyl; G1 stands for -NHCO- or n is 0 or 1; o is 0 or 1; and G2 represents a straight-chain or branched hydrocarbon chain having 1 to 100 carbon atoms, derived from an arylene and/or straight-chain and/or branched and/or cyclic alkylene group, and interrupted once or more by one or more groups of 5 _{to 10} -membered aromatic or non-aromatic heterocycles having up to 4 heteroatoms selected from N, O and S, -SO- or _-SO2- , wherein if a side chain is present, it may be substituted with _-NHCONH2 , -COOH, -OH, _-NH2 , NH- _CNNH2 , sulfonamide, sulfone, sulfoxide or sulfonic acid. Or represents one of the following groups: Rx represents H, _C1 - _C3 -alkyl, or phenyl. 29. The coupling according to claim 28, wherein G2 represents 30. The coupling according to claim 28, wherein L2 is represented by one or both of the following formulas: Where # ¹ refers to the connection point with the sulfur atom of the complex, # ² refers to the connection point with the group ^L1 , ^R22 represents COOH and more than 80% of the bonds to the sulfur atom of the complex exist in one of these two structures based on the total number of bonds between the linker and the complex. 31. The coupling of claim 28, wherein the hydrocarbon chain is interrupted by one of the following groups: Wherein X represents H or a _C1-10 alkyl group that may be optionally substituted with -NHCONH ₂ , -COOH, -OH, NH ₂ , -NH-CNNH ₂ , sulfone, sulfoxide or sulfonic acid. 32. The coupling according to claim 28, wherein the connector has the following formula: in #3 represents the bond that connects to the active substance molecule. #4 represents the bond in the linker. R ¹¹ represents H or NH ₂ ; B represents -[( _CH₂ ) _x -( ^X₄ ) _y ]w-( _CH₂ ) _z- , w = 0 to 20: x = 0 to 5; y = 0 or 1; z = 0 to 5; and X ⁴ represents -O-, -CONH-, or -NHCO- 33. The coupling of claim 28, wherein ^R1 or ^R4 represents -L-#1. 34. The coupling according to claim 28, wherein the coupling has one of the following formulas: in _X1 , _X2 , and _X3 have the same meaning as in claim 1. AK ₁ represents a complex linked by sulfur atoms of the complex; n represents a value from 1 to 20; and L1 represents an optionally branched hydrocarbon group having 1 to 100 carbon atoms, which represents a straight or branched alkyl group having 1 to 100 carbon atoms from an arylene and/or straight and/or branched and/or cyclic alkylene group, and which may be interrupted once or more by one or more groups of ₅ to 10 quintilling aromatic or non-aromatic heterocycles having up to ₄ heteroatoms selected from N, O and S, -SO- or _-SO2- , wherein if a side chain is present, it may be substituted by -NHCONH ₂ , -COOH, -OH, -NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid. Furthermore, SG1 is a 2-8 oligopeptide. 35. The coupling according to claim 34, wherein L1 represents G4 represents 36. The conjugate of claim 34, wherein SG1 is a dipeptide. 37. A conjugate of a complex or its derivative with one or more active substance molecules, wherein the conjugate has the following formula: in _X1 , _X2 , and _X3 have the same meaning as in claim 1. AK ₁ represents a complex linked by sulfur atoms in the complex; n represents a value from 1 to 20; Furthermore, SG1 is a 2-8 oligopeptide; L4 is a single bond or group -(CO) _y -G4-, where y represents 0 or 1, and G4 represents a straight-chain or branched hydrocarbon chain having 1 to 100 carbon atoms, derived from arylene and/or straight-chain and/or branched and/or cyclic alkylene groups, and may be interrupted once or more by one or more groups _-O- , -S-, -SO-, SO2, -NH-, _-CO- , -NHCO-, -CONH-, -NMe-, -NHNH-, -SO2NHNH-, -CONHNH- and one or more heterocycles of 5 to 10 members selected from N, O and S, -SO- or _-SO2- , wherein if a side chain is present, it may be substituted by _-NHCONH2 , -COOH, -OH, _-NH2 , NH- _CNNH2 , sulfonamide, sulfone, sulfoxide or sulfonic acid. 38. The coupling of claim 37, wherein G4 represents 39. The conjugate of claim 37, wherein SG1 is a dipeptide. 40. A conjugate of a complex or its derivative with one or more active substance molecules, wherein the conjugate has one or both of the following formulas: in AK _1A represents a complex linked by sulfur atoms of the complex; n represents a value from 1 to 20; and L1 represents a straight-chain or branched hydrocarbon chain having 1 to 100 carbon atoms, derived from arylene and/or straight-chain and/or branched and/or cyclic alkylene groups, and interrupted once or more by one or more of the groups -O-, -S-, -SO-, _SO₂ , -NH-, -CO-, -CONH-, -NHCO-, -NMe-, -NHNH-, -SO₂NHNH-, -CONHNH- and a _5- to 10-membered aromatic or non-aromatic heterocycle having up to 4 heteroatoms selected from N, O and S, -SO- or _-SO₂- , wherein if a side chain is present, it may be interrupted by _-NHCONH₂ , -COOH, -OH, _-NH₂ , NH- _CNNH₂. The substituted form may contain sulfonamide, sulfone, sulfoxide, or sulfonic acid, and the conjugate or its derivative is a human, humanized, or chimeric monoclonal antibody or its antigen-binding fragment. 41. The coupling of claim 40, wherein L1 represents 42. The conjugate of claim 1, wherein the linker -L- is attached to a cysteine side chain or a cysteine residue and has the following formula: in § represents the bond that connects to the active substance molecule and §§ represents the bond connecting the composite. m is 0, 1, 2, or 3; n is 0, 1, or 2; p is between 0 and 20; and L3 represents in o is 0 or 1; and G3 represents a straight-chain or branched hydrocarbon chain having 1 to 100 carbon atoms, derived from an arylene and/or straight-chain and/or branched and/or cyclic alkylene group, and interrupted once or more by one or more groups of ₅ to 10- _membered aromatic or non-aromatic heterocycles having up to 4 heteroatoms selected from N, O and S, -NMe-, -NHNH-, -SO2NHNH-, -CONHNH-, -SO- or _-SO2- , wherein if a side chain is present, it may be substituted with _-NHCONH2 , -COOH, -OH, sulfone, sulfoxide or sulfonic acid. 43. The coupling according to claim 42, wherein G3 represents 44. The conjugate of claim 42, wherein the linker -L- is attached to a cysteine side chain or cysteine residue and has the following formula: in § represents the bond that connects to the active substance molecule and §§ represents the bond connecting the composite. m is 1; p is 0; n is 0; And L3 represents in o is 0 or 1; and _G3 represents -( _CH₂CH₂O ) _s ( _{CH₂ ) t} (CONH) _u ( _CH₂CH₂O ) _v ( _CH₂ ) _w- , where s, t, v, and w are each independent of each other and range from 0 to 20, and u is 0 or 1. 45. The coupling according to claim 42 or 44, wherein ^R2 or ^R3 represents -L-#1. 46. A conjugate of a complex or its derivative with one or more active substance molecules, wherein the conjugate has one of the following formulas: in _X1 , _X2 , and _X3 have the same meaning as in claim 1. AK ₁ represents a conjugate linked by the sulfur atom of the conjugate; n represents a value from 1 to 20; and L1, L2 and L3 represent a branched, straight-chain or branched hydrocarbon chain having 1 to 100 carbon atoms, derived from arylene and/or straight-chain and/or branched and/or cyclic alkylene groups and interrupted once or more by one or more groups of ₅ to ₁₀ nucleotides of aromatic or non-aromatic heterocycles having up to 4 heteroatoms selected from N, O and S, -SO- or _-SO2- , wherein if a side chain is present, it may be substituted by _-NHCONH2 , -COOH, -OH, _-NH2 , NH- _CNNH2 , sulfonamide, sulfone, sulfoxide or sulfonic acid, and SG1 is a 2-8 oligopeptide. 47. The coupling of claim 46, wherein L1, L2 and L3 represent 48. The conjugate of claim 46, wherein SG1 is a dipeptide. 49. The conjugate of claim 1, wherein the linker -L- is attached to the lysine side chain and has the following formula: -§-(SG) _x -L4-CO-§§ in § represents the bond that connects to the active substance molecule and §§ represents the bond connecting the composite. x represents 0 or 1. SG represents a cleavable group, and L4 represents a single bond or group -(CO) _y -G4-, where y represents 0 or 1, and G4 represents a straight-chain or branched hydrocarbon chain having 1 to 100 carbon atoms, derived from an arylene and/or straight-chain and/or branched and/or cyclic alkylene group, and interrupted once or more by one or more groups of ₅ to 10 nucleotides of aromatic or non-aromatic heterocycles having up to 4 heteroatoms selected from N, O and S or -SO-, wherein if a side chain is present, it may be substituted by -NHCONH ₂ , -COOH, -OH, -NH ₂ , NH-CNNH ₂ , sulfonamide _, sulfone, sulfoxide or sulfonic acid. 50. The coupling of claim 49, wherein G4 represents 51. The conjugate of claim 49, wherein SG is a 2-8 oligopeptide. 52. The conjugate of claim 49, wherein SG is a dipeptide. 53. The coupling of the combination according to claim 49, wherein the coupling has one of the following formulas: in _X1 , _X2 , and _X3 have the same meaning as in claim 1. AK ₂ represents a complex linked by sulfur atoms of the complex; n represents a value from 1 to 20; and L4 represents an optional straight-chain or branched hydrocarbon chain having 1 to 100 carbon atoms, derived from arylene and/or straight-chain and/or branched and/ _or cyclic alkylene groups and interrupted once or more by one or more groups of 5 to ₁₀ nucleotides of aromatic or non-aromatic heterocycles having up to 4 heteroatoms selected from N, O and S, -SO- or _-SO2- , wherein if a side chain is present, it may be substituted by -NHCONH ₂ , -COOH, -OH, -NH ₂ , NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid, and SG1 represents a cleavable group. 54. The coupling of claim 53, wherein L4 represents 55. The conjugate of claim 53, wherein SG1 is a 2-8 oligopeptide. 56. The conjugate of claim 53, wherein SG1 is a dipeptide. 57. The conjugate according to any one of claims 20 to 22, wherein the anti-TWEAKR antibody is an agonist antibody. 58. The coupling according to any one of claims 20 to 22, comprising: Variable heavy chains, which include: a. CDR1 of the heavy chain encoded by the amino acid sequence of the inclusion formula PYPMX (SEQ ID NO: 171), where X is I or M; b. CDR2 of the heavy chain encoded by the amino acid sequence of the inclusion formula YISPSGGXTHYADSVKG (SEQ ID NO: 172), where X is S or K; and c. CDR3 of the heavy chain encoded by the amino acid sequence of inclusion formula GGDTYFDYFDY (SEQ ID NO: 173); and variable light chains, which comprise: d. CDR1 of a light chain encoded by an amino acid sequence of the inclusion formula RASQSISXYLN (SEQ ID NO: 174), wherein X is G or S; e. CDR2 of a light chain encoded by an amino acid sequence (SEQ ID NO: 175) of the inclusion formula XASSLQS, wherein X is Q, A, or N; and f. CDR3 of a light chain encoded by an amino acid sequence (SEQ ID NO: 176) containing the formula QQSYXXPXIT, wherein X at position 5 is T or S, X at position 6 is T or S and X at position 8 is G or F. 59. The coupling according to any one of claims 20 to 22, comprising: a. A variable sequence of the heavy chain as shown in SEQ ID NO: 10 and a variable sequence of the light chain as shown in SEQ ID NO: 9, or b. A variable sequence of the heavy chain as shown in SEQ ID NO: 20 and a variable sequence of the light chain as shown in SEQ ID NO: 19, or c. Variable sequences of the heavy chain as shown in SEQ ID NO: 30 and variable sequences of the light chain as shown in SEQ ID NO: 29, or d. A variable sequence of the heavy chain as shown in SEQ ID NO: 40 and a variable sequence of the light chain as shown in SEQ ID NO: 39, or e. Variable sequences of the heavy chain as shown in SEQ ID NO: 50 and variable sequences of the light chain as shown in SEQ ID NO: 49, or f. A variable sequence of the heavy chain as shown in SEQ ID NO: 60 and a variable sequence of the light chain as shown in SEQ ID NO: 59, or g. Variable sequences of the heavy chain as shown in SEQ ID NO: 70 and variable sequences of the light chain as shown in SEQ ID NO: 69, or h. Variable sequences of the heavy chain as shown in SEQ ID NO: 80 and variable sequences of the light chain as shown in SEQ ID NO: 79, or i. A variable sequence of the heavy chain as shown in SEQ ID NO: 90 and a variable sequence of the light chain as shown in SEQ ID NO: 89, or j. A variable sequence of the heavy chain as shown in SEQ ID NO: 100 and a variable sequence of the light chain as shown in SEQ ID NO: 99, or k. Variable sequences of the heavy chain as shown in SEQ ID NO: 110 and variable sequences of the light chain as shown in SEQ ID NO: 109, or l. Variable sequences of the heavy chain as shown in SEQ ID NO: 120 and variable sequences of the light chain as shown in SEQ ID NO: 119. 60. The conjugate according to any one of claims 20 to 22, wherein the antibody is an IgG antibody. 61. The coupling according to any one of claims 20 to 22, comprising: a. A sequence of the heavy chain as shown in SEQ ID NO: 2 and a sequence of the light chain as shown in SEQ ID NO: 1, or b. A sequence of the heavy chain as shown in SEQ ID NO: 12 and a sequence of the light chain as shown in SEQ ID NO: 11, or c. A sequence of the heavy chain as shown in SEQ ID NO: 22 and a sequence of the light chain as shown in SEQ ID NO: 21, or d. A sequence of the heavy chain as shown in SEQ ID NO: 32 and a sequence of the light chain as shown in SEQ ID NO: 31, or e. A sequence of the heavy chain as shown in SEQ ID NO: 42 and a sequence of the light chain as shown in SEQ ID NO: 41, or f. A sequence of the heavy chain as shown in SEQ ID NO: 52 and a sequence of the light chain as shown in SEQ ID NO: 51, or g. A sequence of the heavy chain as shown in SEQ ID NO: 62 and a sequence of the light chain as shown in SEQ ID NO: 61, or h. A sequence of the heavy chain as shown in SEQ ID NO: 72 and a sequence of the light chain as shown in SEQ ID NO: 71, or i. A sequence of the heavy chain as shown in SEQ ID NO: 82 and a sequence of the light chain as shown in SEQ ID NO: 81, or j. A sequence of the heavy chain as shown in SEQ ID NO: 92 and a sequence of the light chain as shown in SEQ ID NO: 91, or k. A sequence of the heavy chain as shown in SEQ ID NO: 102 and a sequence of the light chain as shown in SEQ ID NO: 101, or l. The sequence of the heavy chain as shown in SEQ ID NO: 112 and the sequence of the light chain as shown in SEQ ID NO: 111. 62. The spindle coupling of claim 1, wherein the coupling has 1 to 10 active substance molecules per conjugate. 63. The spindle coupling of claim 62, wherein the coupling has 2 to 8 active substance molecules per conjugate. 64. A method for preparing the conjugate according to claim 1, wherein a compound of one of the following formula is attached, in the form of its trifluoroacetate, to a cysteine residue of an optionally pre-partially reduced conjugate, wherein the compound is used in a 2 to 12 molar excess relative to the conjugate: Where R represents -H or -COOH, Wherein K represents a straight-chain or branched _C1 - _C6 -alkyl group optionally substituted with _C1 - _C6 -alkoxy or -OH, and _X1 , _X2 , _X3 , SG1, L1, L2, and L3 have the same meaning as in claim 46, and L4 has the same meaning as in claim 49. 65. A method for preparing the conjugate according to claim 64, wherein a compound of one of the following formula is attached to a lysine residue of the conjugate in the form of its trifluoroacetate, wherein the compound is used in a 2 to 12 molar excess relative to the conjugate: _X1 , _X2 , _X3 , SG1 and L4 have the same meaning as in claim 49. 66. A compound with one of the following formulas: Where R represents -H or -COOH, Wherein K represents a straight-chain or branched _C1 - _C6 -alkyl group optionally substituted with _C1 - _C6 -alkoxy or -OH, and _X1 , _X2 , _X3 , SG1, L1, L2, L3 and L4 have the same meaning as in claim 64. 67. Compounds of general formula (III) and their salts: in _X1 represents N, _X2 represents N and _X3 represents C, or _X1 represents CH, _X2 represents C and _X3 represents N; ^R1 represents -L-BINDER, H, or -( _CH2 ) _O-3Z ^, where Z represents -H, ^-NHY3 , ^-OY3 , ^-SY3 , halogen, -CO- ^NY1Y2 , or -CO- ^OY3 . ^Y1 and ^Y2 independently represent H, _NH2 , -( _CH2CH2O ) _O-3- ( _CH2 )O- _3Z ′ or -CH( _CH2W )Z′, and ^Y3 represents H or -( _CH2 )O _{- 3Z} ′, where Z′ represents H, _NH2 , _SO3H , COOH, -NH-CO- _CH2 - _CH2 -CH( _NH2 )COOH or -(CO-NH- ^CHY4 ) _1-3COOH ; where W represents H or OH. ^Y4 independently represents either a straight-chain or branched _C1-6 -alkyl group optionally substituted with -NHCONH ₂ , or an aryl or benzyl group optionally substituted with -NH ₂ ; ^R2 and ^R4 independently represent -L-BINDER, H, -CO-CHY4 ^{- NHY5} , or -( _CH2 ) _0-3Z , or ^R2 and ^R4 together form a pyrrolidine ring representing _-CH2- ^CHR10- or ^-CHR10 - _CH2- , where ^R10 represents -L-BINDER, H, _NH2 , _SO3H , COOH, SH, or OH. Where Z represents -H, halogen, ^-OY3 , ^-SY3 , ^NHY3 , -CO- ^NY1Y2 , or -CO- ^{OY3 . Y1} and ^Y2 independently represent H, _NH2 or -( _CH2 ) _O- 3Z′, and ^Y3 represents H or -( _CH2 ) _O- 3Z′, where Z′ represents H, _SO3H , _NH2 or COOH; ^Y4 independently represents a straight-chain or branched _C1-6 alkyl group optionally substituted with -NHCONH ₂ , or an aryl or benzyl group optionally substituted with -NH ₂ , and ^Y5 represents H or -CO-CHY ⁶ -NH ₂ , wherein ^Y6 represents a straight-chain or branched _C1-6 alkyl group. A represents CO, SO, _SO₂ , _SO₂NH , or CNNH; R ³ represents -L-BINDER or optionally substituted alkyl, aryl, heteroaryl, heteroalkyl, or heterocyclic alkyl; R ⁵ stands for -L-BINDER, H, halogen; Where L represents a linker and BINDER represents a conjugate or a derivative thereof, wherein the conjugate may optionally be linked to multiple compounds. ^R6 and ^R7 independently represent H, optionally fluorinated _C1-10 alkyl, or halogen. R ⁸ represents an optional fluorinated _C1-10 alkyl group; and ^R9 represents H, F, _CH3 , _CF3 , _CH2F , or _CHF2 , and the conjugate or its derivative is a human, humanized, or chimeric monoclonal antibody or its antigen-binding fragment. 68. The compound of claim 67, wherein ^R3 represents -L-BINDER or _C1-10 -alkyl, _C6-10 -aryl or _C6-10 -aralkyl, _C5-10 -heteroalkyl, _C1-10 -alkyl- _OC6-10 -aryl or _C5-10 -heterocyclic alkyl, which may be substituted with 1-3 -OH groups, 1-3 halogen atoms, 1-3 haloalkyl groups each having 1-3 halogen atoms, 1-3 O-alkyl, 1-3 -SH groups, 1-3 -S-alkyl, 1-3 -O-CO-alkyl, 1-3 -O-CO-NH-alkyl, 1-3 -NH-CO-alkyl, 1-3 -NH-CO-NH-alkyl, 1-3 -S(O) _n -alkyl, 1-3 -SO2 _- NH-alkyl, 1-3 -NH-alkyl, 1-3 -N(alkyl) The substitution is ₂ groups, 1-3 _-NH2 groups or 1-3 -( _CH2 ) _0-3Z groups, where Z represents -H, halogen, ^-OY3 , ^-SY3 , ^-NHY3 , -CO- ^NY1Y2 or -CO- ^OY3 , where ^{Y1 and Y2} independently represent H, _NH2 or -( _CH2 ) _0-3Z ′, and ^Y3 represents H, -( _CH2 ) _0-3 -CH( _NHCOCH3 )Z′, -( _CH2 ) _0-3 -CH( _NH2 )Z′ or -( _CH2 ) _0-3Z ′, where Z′ represents H, _SO3H , _NH2 or COOH, where "alkyl" represents _C1-10 -alkyl, and n is 0 or 1. 69. Compounds of general formula (IV) and their salts: in _X1 represents CH, _X2 represents C, and _X3 represents N; ^R1 represents -L-BINDER, H, or -( _CH2 ) _O-3Z ^, where Z represents -H, ^-NHY3 , ^-OY3 , ^-SY3 , halogen, -CO- ^NY1Y2 , or -CO- ^OY3 . ^Y1 and ^Y2 independently represent H, _NH2 , -( _CH2CH2O ) _O-3- ( _CH2 )O- _3Z ′ or -CH( _CH2W )Z′, and ^Y3 represents H or -( _CH2 )O _{- 3Z} ′, where Z′ represents H, _NH2 , _SO3H , COOH, -NH-CO- _CH2 - _CH2 -CH( _NH2 )COOH or -(CO-NH- ^CHY4 ) _1-3COOH ; where W represents H or OH. ^Y4 independently represents either a straight-chain or branched _C1-6 -alkyl group optionally substituted with -NHCONH ₂ , or an aryl or benzyl group optionally substituted with -NH ₂ ; ^R2 and ^R4 independently represent -L-BINDER, H, -CO-CHY4 ^{- NHY5} , or -( _CH2 ) _0-3Z , or ^R2 and ^R4 together form a pyrrolidine ring representing _-CH2- ^CHR10- or ^-CHR10 - _CH2- , where ^R10 represents -L-BINDER, H, _NH2 , _SO3H , COOH, SH, or OH. Where Z represents -H, halogen, ^-OY3 , ^-SY3 , ^NHY3 , -CO- ^NY1Y2 , or -CO- ^{OY3 . Y1} and ^Y2 independently represent H, _NH2 or -( _CH2 ) _O- 3Z′, and ^Y3 represents H or -( _CH2 ) _O- 3Z′, where Z′ represents H, _SO3H , _NH2 or COOH; ^Y4 independently represents a straight-chain or branched _C1-6 alkyl group optionally substituted with -NHCONH ₂ , or an aryl or benzyl group optionally substituted with -NH ₂ , and ^Y5 represents H or -CO-CHY ⁶ -NH ₂ , wherein ^Y6 represents a straight-chain or branched _C1-6 alkyl group. A represents CO, SO, _SO₂ , _SO₂NH , or CNNH; R ³ represents -L-BINDER or optionally substituted alkyl, aryl, heteroaryl, heteroalkyl, or heterocyclic alkyl; R ⁵ stands for -L-BINDER, H, halogen; Where L represents a linker and BINDER represents a conjugate or a derivative thereof, wherein the conjugate may optionally be linked to multiple compounds. ^R6 and ^R7 independently represent H, optionally fluorinated _C1-10 alkyl, or halogen. R ⁸ represents an optional fluorinated _C1-10 alkyl group; and ^R9 represents H, F, _CH3 , _CF3 , _CH2F , or _CHF2 , and the conjugate or its derivative is a human, humanized, or chimeric monoclonal antibody or its antigen-binding fragment. 70. The compound of claim 69, wherein ^R3 represents -L-BINDER or _C1-10 -alkyl, _C6-10 -aryl, _C6-10 -aralkyl, C5-10-heteroalkyl, _C1-10 -alkyl- _OC6-10 -aryl- or _C5-10 -heterocyclic alkyl, which may be substituted with _1-3 -OH groups, 1-3 halogen atoms, 1-3 haloalkyl groups each having 1-3 halogen atoms, 1-3 O-alkyl groups, 1-3 -SH groups, 1-3 -S-alkyl groups, 1-3 -O-CO-alkyl groups, 1-3 -O-CO-NH-alkyl groups, 1-3 -NH-CO-alkyl groups, 1-3 -NH-CO-NH-alkyl groups, 1-3 -S(O) _n -alkyl groups, 1-3 -SO2-NH-alkyl groups, _1-3 -NH-alkyl groups, 1-3 -N(alkyl) The substitution is ₂ groups, 1-3 _-NH2 groups or 1-3 -( _CH2 ) _0-3Z groups, where Z represents -H, halogen, ^-OY3 , ^-SY3 , ^-NHY3 , -CO- ^NY1Y2 or -CO- ^OY3 , where ^{Y1 and Y2} independently represent H, _NH2 or -( _CH2 ) _0-3Z ′, and ^Y3 represents H, -( _CH2 ) _0-3 -CH( _NHCOCH3 )Z′, -( _CH2 ) _0-3 -CH( _NH2 )Z′ or -( _CH2 ) _0-3Z ′, where Z′ represents H, _SO3H , _NH2 or COOH, where "alkyl" represents _C1-10 -alkyl, and n is 0 or 1. 71. The compound of claim 69, wherein Z represents Cl or Br. 72. _The compound according to claim 69, wherein ^R1 represents -( _CH2 ) _O- 3Z, wherein Z represents -CO ^{- NY1Y2} , wherein ^Y2 represents -( _CH2CH2O ) _O-3- ( _CH2 )O _-3Z ′, and ^Y1 represents H, _NH2 or -( _CH2CH2O ) _O-3- ( _CH2 ) _{O- 3Z} ′. 73. The compound according to claim 72, wherein ^Y1 represents H, ^Y2 represents -( _CH2CH2O ) _{3- CH2CH2Z ′ and} Z′ represents -COOH. 74. The compound according to claim 72, wherein ^Y1 represents H, ^Y2 represents _{-CH2CH2Z ′} and Z′ represents -( ^CONHCHY4 ) _2COOH . 75. The compound of claim 74, wherein ^Y4 represents a straight-chain or branched _C1-6 -alkyl group optionally substituted with _-NHCONH2 . 76. The compound of claim 75, wherein at least one of the ^Y4 groups is selected from isopropyl and _-CH3 . 77. The compound according to claim 72, wherein ^Y1 represents H, ^Y2 represents _-CH2CH2Z ′, Z′ represents ^{-CONHCHY4COOH} _, and ^Y4 represents an aryl or benzyl group optionally substituted with _-NH2 . 78. The compound of claim 77, wherein ^Y4 represents an aminobenzyl group. 79. The compound according to claim 69, wherein ^R2 represents -( _CH2 ) _O-3Z and Z represents ^-SY3 . 80. The compound of claim 69, wherein R ⁴ represents -CO-CHY ⁴ -NHY ⁵ and Y ⁵ represents H, wherein Y ⁴ represents a straight-chain or branched _C1-6 -alkyl optionally substituted with -NHCONH ₂ . 81. The compound of claim 69, wherein R ⁴ represents -CO-CHY ⁴ -NHY ⁵ and Y ⁵ represents -CO-CHY ⁶ -NH ₂ , wherein Y ⁴ represents a straight-chain or branched _C1-6 -alkyl optionally substituted with -NHCONH ₂ . 82. The compound according to any one of claims 69 to 81, wherein the compound has one of the following formula: N-(3-aminopropyl)-N-{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide; N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide; (2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-N-methylbutyramide; N-(3-aminopropyl)-N-{(1S)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide; S-[1-(2-{[2-({(2S)-2-amino-4-[{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]amino}-2-oxoethyl)-2,5-dioxopyrrolidone-3-yl]-L-cysteine; N-{(2S)-2-amino-4-[{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl-L-alanyl-N-[4-(3-{[(2R)-2-amino-2-carboxyethyl]thioalkyl}-2,5-dioxopyrrolidine-1-yl)phenyl]-N ⁵ -carbamoyl-L-guanineamide; S-(1-{2-[(N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl)amino]ethyl}-2,5-dioxopyrrolidine-3-yl)-L-cysteine; N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]thioalkyl}-2,5-dioxopyrrolidone-1-yl)hexanoyl]-L-valine-N5-carbamoyl-L-guanine- ^N6 -{(2S)-2-amino-4-[{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-L-lysine; S-[1-(2-{[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]amino}-2-oxoethyl)-2,5-dioxopyrrolidin-3-yl]-L-cysteine; S-(2-{[2-({(2S)-2-amino-4-[{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]amino}-2-oxoethyl)-L-cysteine; S-{1-[6-(2-{(2S)-2-amino-4-[{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}hydrazino)-6-oxohexyl]-2,5-dioxopyrrolidine-3-yl}-L-cysteine; N-[19-(3(R/S)-{[(2R)-2-amino-2-carboxyethyl]thioalkyl}-2,5-dioxopyrrolidine-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-aza-nonadecano-1-acyl]-R/S-{2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}homocysteine; S-{(3R/S)-1-[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]-2,5-dioxopyrrolidine-3-yl}-L-cysteine; N-[19-(3(R/S)-{[(2R)-2-amino-2-carboxyethyl]thioalkyl}-2,5-dioxopyrrolidone-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-aza-nonadecano-1-acyl]-R/S-{2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}homocysteine; S-[(3R/S)-1-(2-{[6-({2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}thioalkyl)hexanoyl]amino}ethyl)-2,5-dioxopyrrolidin-3-yl]-L-cysteine; S-{1-[2-({[(1R,3S)-3-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)cyclopentyl]carbonyl}amino)ethyl]-2,5-dioxopyrrolidin-3-yl}-L-cysteine; S-(2-{[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]amino}-2-oxoethyl)-L-cysteine; ^N6- (N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl) ^-N2- {N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]thioalkyl}-2,5-dioxopyrrolo-1-yl)hexanoyl]-L-valine-L-alanyl}-L-lysine; N-[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]-L-glutamine; N ⁶ -(N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl)-L-lysine; N-(3-aminopropyl)-N-{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}acetamide; N-(3-aminopropyl)-N-{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}-2-methoxyacetamide; N-(3-aminopropyl)-N-{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}-2,4-difluorobenzamide; N-(3-aminopropyl)-N-{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}-4-methylbenzamide; N-(3-aminopropyl)-N-{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}-2-ethoxyacetamide; N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}-3,3,3-trifluoropropionamide; N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}-4-fluorobenzamide; N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}acetamide; N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}-4-(trifluoromethyl)benzamide; N-(3-aminopropyl)-N-{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}-2-ethoxyacetamide; N-(3-aminopropyl)-N-{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}-2-ethoxyacetamide; (2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyric acid; (2S)-2-amino-N-(2-aminoethyl)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyramide; 4-[(2-{[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]amino}-2-oxoethyl)amino]-3-{[(2R)-2-amino-2-carboxyethyl]thioalkyl}-4-oxobutyric acid; 4-[(2-{[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]amino}-2-oxoethyl)amino]-2-{[(2R)-2-amino-2-carboxyethyl]thioalkyl}-4-oxobutyric acid; N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanine; N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-L-serine; N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-L-alanine; N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}glycine; N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}-4-methylbenzamide; N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}-4-(methylthioalkyl)benzamide; (2S)-N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}-2-hydroxypropionamide; N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}-2-(methylthioalkyl)acetamide; (2S)-N-(3-aminopropyl)-N-{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}-2-hydroxypropionamide; 4-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}amino]-4-oxobutyrate methyl ester; 4-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}amino]-4-oxobutyric acid; (2R)-22-[(3R/S)-3-{[(2R)-2-amino-2-carboxyethyl]thioalkyl}-2,5-dioxopyrrolidine-1-yl]-2-[({2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}thioalkyl)methyl]-4,20-dioxo-7,10,13,16-tetraoxa-3,19-diazacosanodecane-1-acid; 4-Amino-N-(3-aminopropyl)-N-{(1R)-1-[4-benzyl-1-(2,5-difluorophenyl)-1H-pyrazol-3-yl]-2,2-dimethylpropyl}benzamide; N-acetyl-S-{2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}-L-cysteine; N-acetyl-S-[2-([3-(L-alanylamino)propyl]{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}amino)-2-oxoethyl]-L-cysteine; (2S)-N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}tetrahydrofuran-2-carboxamide; 3-({2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}thioalkyl)propionic acid; S-{2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}homocysteine; 4-Amino-N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}benzamide; 4-[(2-{[(2R)-2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)-2-carboxyethyl]amino}-2-oxoethyl)amino]-3-{[(2R)-2-amino-2-carboxyethyl]thioalkyl}-4-oxobutyric acid; 4-[(2-{[(2R)-2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)-2-carboxyethyl]amino}-2-oxoethyl)amino]-2-{[(2R)-2-amino-2-carboxyethyl]thioalkyl}-4-oxobutyric acid. 83. A pharmaceutical composition comprising a conjugate according to any one of claims 1 to 63 or a compound according to any one of claims 69 to 82, and an inert, non-toxic, pharmaceutically acceptable adjuvant. 84. A conjugate of any one of claims 1 to 63 or a compound of any one of claims 69 to 82 used in methods of treating and/or preventing disease. 85. A conjugate of any one of claims 1 to 63 or a compound of any one of claims 69 to 82 used in a method for treating diseases of hyperplasia and/or angiogenesis. 86. The coupling of any one of claims 1 to 63, wherein ^R3 represents -L-#1 or a substituted alkyl, aryl or heteroaryl group. 87. The coupling of claim 86, wherein ^R3 represents -L-#1 or _C1-10 -alkyl, _C6-10 -aryl, _C6-10 -aralkyl, or _C5-10 -heteroalkyl, which may be substituted with -OH, O-alkyl, SH, S-alkyl, O-CO-alkyl, O-CO-NH-alkyl, NH-CO-alkyl, NH-CO-NH-alkyl, S(O) _n -alkyl, _SO2 -NH-alkyl, NH-alkyl, N(alkyl) ₂ , _NH2 , or -( _CH2 ) _O-3Z , wherein Z represents ^-H , halogen, ^-OY3 , ^-SY3 , ^{-NHY3, -CO-NY1Y2 ,} or -CO- ^OY3 , wherein ^Y1 and ^Y2 independently represent H, _NH2 , or -( _CH2 ) _O- 3Z′, and ^Y3 represents H or -( _CH2 ) _0-3 Z′, where Z′ represents H, _SO3H , _NH2 or COOH, and “alkyl” represents _C1-10 -alkyl. 88. The compound according to any one of claims 69 to 82, wherein ^R3 represents -L-BINDER or a substituted alkyl, aryl or heteroaryl group. 89. The compound according to claim 88, wherein R3 represents -L-BINDER or _C1-10 -alkyl, _C6-10 -aryl, _C6-10 -aralkyl, or _C5-10 -heteroalkyl, which may be substituted with -OH, O-alkyl, SH, S-alkyl, O-CO-alkyl, O-CO-NH-alkyl, NH-CO-alkyl, NH-CO-NH-alkyl, S(O) _n -alkyl, _SO2 -NH-alkyl, NH-alkyl, N(alkyl) ₂ , _NH2 , or -( _CH2 ) _O-3Z , wherein Z represents -H, halogen, ^-OY3 , ^-SY3 , ^-NHY3 , -CO- ^{NY1Y2 , or -CO-OY3 ,} wherein ^Y1 and ^Y2 independently represent H, _NH2 , or -( _CH2 ) _O- 3Z′, and ^Y3 represents H or -( _CH2 ) _0-3 Z′, where Z′ represents H, _SO3H , _NH2 or COOH, and “alkyl” represents _C1-10 -alkyl. 90. Use in an effective amount of at least one conjugate according to any one of claims 1 to 63 or a compound according to any one of claims 69 to 82 in the preparation of a medicament for the treatment and/or prevention of hyperplastic and/or angiogenic diseases in humans and animals. 91. An antibody conjugate according to one of the following formulas, where n is a value from 1 to 20 and AK _1A and AK _2A represent antibodies: If the ADC is attached to the antibody via a group, it can also be replaced by at least part of the form of hydrolyzed open-chain succinamide attached to the antibody.